In vivo and in vitro olefin cyclopropanation catalyzed by heme enzymes

ABSTRACT

The present invention provides methods for catalyzing the conversion of an olefin to any compound containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product comprising providing an olefinic substrate, a diazo reagent, and a heme enzyme; and admixing the components in a reaction for a time sufficient to produce a cyclopropanation product. In other aspects, the present invention provides heme enzymes including variants and fragments thereof that are capable of carrying out in vivo and in vitro olefin cyclopropanation reactions. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of PCT/US2013/063577, filed Oct. 4, 2013, which application claims priority to U.S. Provisional Application No. 61/711,640, filed Oct. 9, 2012, U.S. Provisional Application No. 61/740,247, filed Dec. 20, 2012, U.S. Provisional Application No. 61/784,917, filed Mar. 14, 2013, U.S. Provisional Application No. 61/838,167, filed Jun. 21, 2013, U.S. Provisional Application No. 61/815,997, filed Apr. 25, 2013, U.S. Provisional Application No. 61/818,329, filed May 1, 2013, and U.S. Provisional Application No. 61/856,493, filed Jul. 19, 2013. The disclosures of each of these applications and International Application No. PCT/US2013/63428, filed Oct. 4, 2013, are hereby incorporated by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under DE-FG02-06ER15762/T-105789 awarded by the Department of Energy and under 1F32EB015846-01. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

A considerable challenge in modern synthetic chemistry is the selective direct functionalization of unactivated carbon-hydrogen (C—H) bonds and carbon-carbon (C═C) double bonds (e.g., olefins) (R. G. Bergman, Nature 446, 391 (2007); H. Pellissier, Tetrahedron 64, 7041 (2008)). Adapting asymmetric catalytic processes to these reactions has important consequences in the stereoselective and regioselective elaboration of molecules for natural product and pharmaceutical synthesis. In recent years, much success has been achieved in the development of catalysts for the select addition of oxygen into molecules (M. S. Chen et al., Science 318, 783 (2007)). More challenging is the direct introduction of new carbon-carbon centers into complex structures. A contemporary catalytic approach uses metallocarbenoid intermediates that transfer a reactive carbene into select C—H and C═C bonds, creating new asymmetric highly substituted carbon centers and cyclopropanes, respectively (H. M. L. Davies et al., Chemical Reviews 103, 2861 (2003)). However, the most successful catalysts to date often utilize expensive and possibly toxic transition metal complexes, with dirhodium species marking representative examples. Notably, high yield, regioselectivity, and stereoselectivity in these systems remains difficult to achieve and many of these catalysts are hampered by harsh reaction conditions including high temperature and organic solvents.

The asymmetric cyclopropanation of olefins with high-energy carbene precursors is a hallmark reaction that generates up to 3 stereogenic centers in a single step to make the important cyclopropane motif, featured in many natural products and therapeutic agents (H. Lebel et al., Chemical Reviews 103, 977 (2003)). Limited to using physiologically accessible reagents, Nature catalyzes intermolecular cyclopropane formation through wholly different strategies, typically involving olefin addition to the methyl cation of S-adenosyl methionine or through cyclization of dimethylallyl pyrophosphate-derived allylic carbenium ions (L. A. Wessjohann et al., Chemical Reviews 103, 1625 (2003)). As a result, the diverse cyclopropanation products that can be formed by metallocarbene chemistry cannot be readily accessed by engineering natural cyclopropanation enzymes. As such, there is a need in the art for novel reagents and catalytic schemes that are capable of creating the cyclopropane motif with high yield, regioselectivity, and stereoselectivity, but without the toxicity and harsh reaction conditions associated with current approaches. The present invention satisfies this need by providing novel iron-heme-containing enzyme catalysts for producing cyclopropanation products in vitro and in vivo, and offers related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides methods for catalyzing the conversion of an olefin to any compound (e.g., any intermediate or final compound) containing one or more cyclopropane functional groups using heme enzymes.

In certain aspects, the present invention provides a method for producing a cyclopropanation product, the method comprising:

-   -   (a) providing an olefinic substrate, a diazo reagent, and a heme         enzyme; and     -   (b) admixing the components of step (a) in a reaction for a time         sufficient to produce a cyclopropanation product.

In some embodiments, the cyclopropanation product is a compound according to Formula I:

wherein:

-   -   R¹ is independently selected from the group consisting of H,         optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀         aryl, optionally substituted 6- to 10-membered heteroaryl, halo,         cyano, C(O)OR^(1a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and         Si(R⁸)₃;     -   R² is independently selected from the group consisting of H,         optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀         aryl, optionally substituted 6- to 10-membered heteroaryl, halo,         cyano, C(O)OR^(2a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and         Si(R⁸)₃;     -   wherein:         -   R^(1a) and R^(2a) are independently selected from the group             consisting of H, optionally substituted C₁₋₁₈ alkyl and             -L-R^(C), wherein         -   each L is selected from the group consisting of a bond,             —C(R^(L))₂—, and —NR^(L)—C(R^(L))₂—,         -   each R^(L) is independently selected from the group             consisting of H, C₁₋₆ alkyl, halo, —CN, and —SO₂, and         -   each R^(C) is selected from the group consisting of             optionally substituted C₆₋₁₀ aryl, optionally substituted 6-             to 10-membered heteroraryl, and optionally substituted 6- to             10-membered heterocyclyl; and     -   R³, R⁴, R⁵, and R⁶ are independently selected from the group         consisting of H, C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl,         optionally substituted C₆₋₁₀ aryl, optionally substituted C₁-C₆         alkoxy, halo, hydroxy, cyano, C(O)N(R⁷)₂, NR⁷C(O)R⁸, C(O)R⁸,         C(O)OR⁸, and N(R⁹)₂,     -   wherein:         -   each R⁷ and R⁸ is independently selected from the group             consisting of H, optionally substituted C₁₋₁₂ alkyl,             optionally substituted C₂₋₁₂ alkenyl, and optionally             substituted C₆₋₁₀ aryl; and         -   each R⁹ is independently selected from the group consisting             of H, optionally substituted C₆₋₁₀ aryl, and optionally             substituted 6- to 10-membered heteroaryl, or two R⁹             moieties, together with the nitrogen atom to which they are             attached, can form 6- to 18-membered heterocyclyl;         -   or R³ forms an optionally substituted 3- to 18-membered ring             with R⁴;         -   or R⁵ forms an optionally substituted 3- to 18-membered ring             with R⁶;         -   or R³ or R⁴ forms a double bond with R⁵ or R⁶;         -   or R³ or R⁴ forms an optionally substituted 5- to 6-membered             ring with R⁵ or R⁶.

In certain embodiments, R¹ is C(O)O-LR^(c); R² is selected from the group consisting of H and optionally substituted C₆₋₁₀ aryl; and R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H, optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, optionally substituted C₆₋₁₀ aryl and halo, or R³ forms an optionally substituted 3- to 18-membered ring with R⁴; or R⁵ forms an optionally substituted 3- to 18-membered ring with R⁶.

In certain other embodiments, the cyclopropanation product is a compound according to Formula II:

-   -   wherein R^(1a) is C₁₋₆ alkyl and R² is selected from the group         consisting of H and optionally substituted C₆₋₁₀ aryl.

In some instances, R² is H. In other instances, the cyclopropanation product is selected from the group consisting of:

wherein:

-   -   X¹ is selected from the group consisting of H, optionally         substituted C₁₋₆ alkyl, haloC₁₋₆ alkyl, optionally substituted         C₁₋₆ alkoxy, optionally substituted C₁₋₆ alkylthio, C₁₋₆         alkylsilyl, halo, and cyano;     -   X² is selected from the group consisting of H, chloro, and         methyl;     -   X³ is selected from the group consisting of H, methyl, halo, and         CN;     -   each X⁴ is independently halo;     -   each X⁵ is independently selected from the group consisting of         methyl and halo,     -   X⁶ is selected from the group consisting of halo, optionally         substituted C₁₋₆ alkyl, and optionally substituted C₁₋₆ alkoxy;     -   X⁷ is selected from the group consisting of H, methyl, and halo;     -   X⁸ is selected from the group consisting of H, halo, and         optionally substituted C₁₋₆ alkyl;     -   X⁹ is selected from the group consisting of H, halo, optionally         substituted C₁₋₆ alkyl, C(O)O—(C₁₋₆ alkyl), C(O)—N(C₁₋₆ alkyl)₂,         and cyano; and     -   Z¹, Z², and Z³ are independently selected from the group         consisting of H, halo, optionally substituted C₁₋₆ alkyl, and         optionally substituted C₆₋₁₀ aryl; or     -   Z¹ and Z² are taken together to form an optionally substituted         5- to 6-membered cycloalkyl or heterocyclyl group.

In certain embodiments, the method further comprises converting the cyclopropanation product to a compound according to Formula III:

wherein:

-   -   L is selected from the group consisting of a bond, —C(R^(L))₂—,         and —NR^(L)—C(R^(L))₂—,     -   each R^(L) is independently selected from the group consisting         of H, —CN, and —SO₂, and     -   R^(1c) is selected from the group consisting of optionally         subsistuted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered         heteroraryl, and optionally substituted 6- to 10-membered         heterocyclyl.

In some instances, L is selected from the group consisting of a bond, —CH₂—, —CH(CN)—, and —N(SO₂)—CH₂. In other instances, the moiety L-R^(1c) has a structure selected from the group consisting of:

wherein:

-   -   each Y¹ is independently selected from the group consisting of         optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆         alkenyl, optionally substituted C₂₋₆ alkynyl, phenyl, and         (phenyl)C₁₋₆ alkoxy;     -   each Y² is independently selected from the group consisting of         halo, optionally substituted C₁₋₆ alkyl, optionally substituted         C₂₋₆ alkenyl, optionally substituted C₁₋₆ alkoxy, and nitro;     -   each Y³ is independently optionally substituted C₁₋₆ alkyl;     -   each Y⁴ is independently selected from the group consisting of         optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆         alkenyl, optionally substituted C₂₋₆ alkynyl, C₆₋₁₀ aryl-C₁₋₆         alkyl, furfuryl, C₁₋₆ alkoxy, (C₂₋₆ alkenyl)oxy, C₁₋₁₂ acyl, and         halo;     -   Y⁵ is selected from the group consisting of optionally         substituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, and         halo,     -   the subscript m is an integer from 1 to 3,     -   the subscript n is an integer from 1 to 5,     -   the subscript p is an integer from 1 to 4,     -   the subscript q is an integer from 0 to 3,     -   and the wavy line at the left of the structure represents the         point of connection between the moiety -L—R^(1c) and the rest of         the compound according to Formula III.

In yet other instances, the compound according to Formula III is resmethrin.

In certain embodiments, the cyclopropanation product is a compound having a structure according to the formula:

wherein:

-   -   R^(1a) is optionally substituted C₁₋₆ alkyl, and     -   R⁵ and R⁶ are independently selected from the group consisting         of H, optionally substituted C₁₋₆ alkyl, optionally substituted         C₆₋₁₀ aryl, C(O)N(R⁷)₂, C(O)OR⁸ and NR⁷C(O)R⁸.

In some instances, the cyclopropanation product has a structure selected from the group consisting of:

In certain instances, the method further comprises converting the cyclopropanation product to a compound selected from the group consisting of milnacipran, levomilnacipran, bicifadine, and 1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0]hexane.

In some embodiments, the olefinic substrate is selected from the group consisting of an alkene, a cycloalkene, and an arylalkene. In certain instances, the olefinic substrate comprises an arylalkene. In some instances, the arylalkene is a styrene. In other instances, the styrene has the formula:

-   -   wherein R³ is selected from the group consisting of H,         optionally substituted C₁-C₆ alkyl, optionally substituted C₁-C₆         alkoxy, C(O)N(R⁷)₂, C(O)OR⁸, N(R⁹)₂, halo, hydroxy, and cyano;     -   R⁵ and R⁶ are independently selected from the group consisting         of H, optionally substituted C₁₋₆ alkyl, and halo;     -   R¹⁰ is selected from the group consisting of optionally         substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy,         halo, and haloalkyl; and     -   the subscript r is an integer from 0 to 2.

In some embodiments, the diazo reagent has a structure according to the formula:

wherein:

-   -   R¹ is independently selected from the group consisting of H,         optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀         aryl, optionally substituted 6- to 10-membered heteroaryl, halo,         cyano, C(O)OR^(1a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and         Si(R⁸)₃; and     -   R² is independently selected from the group consisting of H,         optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀         aryl, optionally substituted 6- to 10-membered heteroaryl, halo,         cyano, C(O)OR^(2a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and         Si(R⁸)₃;     -   wherein         -   R^(1a) and R^(2a) are independently selected from the group             consisting of H, optionally substituted C₁₋₁₈ alkyl and             -L-R^(C), wherein         -   each L is selected from the group consisting of a bond,             —C(R^(L))₂—, and —NR^(L)—C(R¹)₂—,         -   each R^(L) is independently selected from the group             consisting of H, C₁₋₆ alkyl, halo, —CN, and —SO₂, and         -   each R^(C) is selected from the group consisting of             optionally substituted C₆₋₁₀ aryl, optionally substituted 6-             to 10-membered heteroraryl, and optionally substituted 6- to             10-membered heterocyclyl; and         -   each R⁷ and R⁸ is independently selected from the group             consisting of H, optionally substituted C₁₋₁₂ alkyl,             optionally substituted C₂₋₁₂ alkenyl, and optionally             substituted C₆₋₁₀ aryl.

In certain embodiments, the diazo reagent is selected from the group consisting of an α-diazoester, an α-diazoamide, an α-diazonitrile, an α-diazoketone, an α-diazoaldehyde, and an α-diazosilane.

In certain instances, the diazo reagent has a formula selected from the group consisting of:

-   -   wherein         -   R^(1a) is selected from the group consisting of H and             optionally substituted C₁-C₆ alkyl; and         -   each R⁷ and R⁸ is independently selected from the group             consisting of H, optionally substituted C₁₋₁₂ alkyl,             optionally substituted C₂₋₁₂ alkenyl, and optionally             substituted C₆₋₁₀ aryl.

In certain other instances, the diazo reagent is selected from the group consisting of diazomethane, ethyl diazoacetate, and (trimethylsilyl)diazomethane. In yet other instances, the diazo reagent has the formula:

In particular embodiments, the cyclopropanation product has a formula selected from the group consisting of:

In some embodiments, the method is carried out in vitro. In certain instances, the reaction further comprises a reducing agent. In other embodiments, the heme enzyme is localized within a whole cell and the method is carried out in vivo. In some embodiments, the method is carried out under anaerobic conditions.

In some embodiments, the method produces a plurality of cyclopropanation products. In certain instances, the plurality of cyclopropanation products has a Z:E ratio of from 1:99 to 99:1. In some instances, the plurality of cyclopropanation products has a % ee_(z) of from about −90% to about 90%. In other instances, the plurality of cyclopropanation products has a % ee_(E) of from about −90% to about 90%. In some instances, the cyclopropanation reaction is at least 30% to at least 90% diasteroselective. In other instances, the cyclopropanation reaction is at least 30% to at least 90% enantioselective.

In some embodiments, the heme enzyme is expressed in a bacterial, archaeal, or fungal host organism.

In certain embodiments, the heme enzyme is a fragment thereof comprising the heme domain. In particular embodiments, the heme enzyme is an engineered heme enzyme such as a heme enzyme variant comprising a mutation at the axial position of the heme coordination site. In some instances, the mutation is a substitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the axial position. In certain instances, the mutation is a substitution of Cys with Ser at the axial position. In other embodiments, the heme enzyme variant is a chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing proteins.

In particular embodiments, the heme enzyme is a cyclochrome P450 enzyme or a variant thereof. In preferred embodiments, the cyclochrome P450 enzyme is a P450 BM3 enzyme or a variant thereof.

In certain embodiments, the cyclochrome P450 enzyme variant comprises a mutation at the axial position of the heme coordination site. In some instances, the mutation is a substitution of Cys with Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the axial position. In certain instances, the mutation is a substitution of Cys with Ser at the axial position. In other embodiments, the cyclochrome P450 variant is a chimeric protein comprising recombined sequences or blocks of amino acids from at least two, three, or more different P450 enzymes (e.g., CYP102A1 (P450 BM3), CYP102A2, and CYP102A3).

In some embodiments, the P450 BM3 enzyme comprises the amino acid sequence set forth in SEQ ID NO:1. In particular embodiments, the P450 BM3 enzyme variant comprises a C400X mutation at the axial position in SEQ ID NO:1, wherein X is any amino acid other than Cys. In other embodiments, the P450 BM3 enzyme variant comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen of the following amino acid substitutions in SEQ ID NO:1: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K.

In some embodiments, the P450 BM3 enzyme variant comprises at least one, two, or all three of the following amino acid substitutions in SEQ ID NO:1: 1263A, A328G, and a T438 mutation. In certain instances, the T438 mutation is T438A, T438S, or T438P.

In some embodiments, the P450 BM3 enzyme variant comprises from one to five alanine substitutions in the active site of SEQ ID NO:1. In certain instances, the active site alanine substitutions are selected from the group consisting of L75A, M177A, L181A, I263A, L437A, and a combination thereof.

In particular embodiments, the P450 BM3 enzyme variant comprises a T268A mutation and/or a C400X mutation in SEQ ID NO:1, wherein X is any amino acid other than Cys.

In some embodiments, the heme enzyme comprises a fragment of the cytochrome P450 enzyme or variant thereof. In certain instances, the fragment comprises the heme domain of the cyclochrome P450 enzyme or variant thereof.

In particular embodiments, the heme enzyme is a P450 enzyme variant selected from Tables 4, 5A, and 5B.

In other aspects, the present invention provides a heme enzyme or a fragment thereof that can cyclopropanate an olefinic substrate.

In particular embodiments, the heme enzyme is an engineered heme enzyme such as a heme enzyme variant comprising a mutation at the axial position of the heme coordination site. In some instances, the mutation is a substitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the axial position. In certain instances, the mutation is a substitution of Cys with Ser at the axial position. In other embodiments, the heme enzyme variant is a chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing proteins.

In some embodiments, the heme enzyme variant is isolated and/or purified. In other embodiments, the heme enzyme variant is a cyclochrome P450 enzyme variant. In preferred embodiments, the cyclochrome P450 enzyme variant is a P450 BM3 enzyme variant.

In certain embodiments, the cyclochrome P450 enzyme variant comprises a mutation at the axial position of the heme coordination site. In some instances, the mutation is a substitution of Cys with Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the axial position. In certain instances, the mutation is a substitution of Cys with Ser at the axial position. In other embodiments, the cyclochrome P450 variant is a chimeric protein comprising recombined sequences or blocks of amino acids from at least two, three, or more different P450 enzymes (e.g., CYP102A1, CYP102A2, and CYP102A3).

In certain embodiments, the P450 BM3 enzyme variant comprises at least one mutation in the amino acid sequence set forth in SEQ ID NO:1. In particular embodiments, the P450 BM3 enzyme variant comprises a C400X mutation at the axial position in SEQ ID NO:1, wherein X is any amino acid other than Cys. In other embodiments, the P450 BM3 enzyme variant comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen of the following amino acid substitutions in SEQ ID NO:1: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K.

In some embodiments, the P450 BM3 enzyme variant comprises at least one, two, or all three of the following amino acid substitutions in SEQ ID NO:1: 1263A, A328G, and a T438 mutation. In certain instances, the T438 mutation is T438A, T438S, or T438P.

In some embodiments, the P450 BM3 enzyme variant comprises from one to five alanine substitutions in the active site of SEQ ID NO:1. In certain instances, the active site alanine substitutions are selected from the group consisting of L75A, M177A, L181A, I263A, L437A, and a combination thereof.

In particular embodiments, the P450 BM3 enzyme variant comprises a T268A mutation and/or a C400X mutation in SEQ ID NO:1, wherein X is any amino acid other than Cys.

In some embodiments, the heme enzyme variant fragment comprises the heme domain thereof. In particular embodiments, the heme enzyme variant is a P450 enzyme variant selected from Tables 4, 5A, and 5B.

In other embodiments, the heme enzyme variant has a higher total turnover number (TTN) compared to the wild-type sequence. In certain instances, the heme enzyme variant has a TTN greater than about 100.

In some instances, the heme enzyme variant produces a plurality of cyclopropanation products having a Z:E ratio of from 1:99 to 99:1. In some instances, the heme enzyme variant produces a plurality of cyclopropanation products having a % ee_(z) of at least −90% to at least 90%. In other instances, the heme enzyme variant produces a plurality of cyclopropanation products having a % ee_(E) of at least −90% to at least 90%. In some instances, the heme enzyme variant produces a plurality of cyclopropanation products having at least 30% to at least 90% diasteroselectivity. In other instances, the heme enzyme variant produces a plurality of cyclopropanation products having at least 30% to at least 90% enantioselectivity. In yet other instances, the heme enzyme variant is in lyophilized form.

In further aspects, the present invention provides a cell expressing a heme enzyme described herein. In certain embodiments, the cell is a bacterial cell or a yeast cell.

In yet other aspects, the present invention provides an expression vector comprising a nucleic acid sequence encoding a heme enzyme described herein. In related aspects, the present invention provides a cell comprising the expression vector. In certain embodiments, the cell is a bacterial cell or a yeast cell.

Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the canonical mode of reactivity of cytochrome P450s. (Left): monooxygenation of olefins and C—H bonds to epoxides and alcohols catalyzed by the ferryl porphyrin radical intermediate (Compound I). (Right): Artificial mode of formal carbene transfer activity of cytochrome P450s utilizing diazoester reagents as carbene precursors.

FIG. 2 illustrates the absolute stereoselectivity of select P450_(BM3) cyclopropanation catalysts. Reaction conditions: 20 μM catalyst, 30 mM styrene, 10 mM EDA, 10 mM Na₂S₂O₄, under argon in aqueous potassium phosphate buffer (pH 8.0) and 5% MeOH cosolvent for 2 hours at 298 K. Enzyme loading is 0.2 mol % with respect to EDA. The structures of each product stereoisomer are shown above the reaction gas chromatograms.

FIG. 3 illustrates the effect of styrene concentration on cyclopropane yield.

FIG. 4 illustrates the effect of P450 (H2A10) concentration on cyclopropane yield.

FIGS. 5A-B illustrate the initial velocities plot for variant 9-10A-TS-F87V-T268A (C3C)_(heme). (FIG. 5A) EDA concentration was varied at a saturating concentration of styrene (30 mM). (FIG. 5B) Styrene concentration was varied at a fixed concentration of EDA (20 mM). Initial rates were computed as the slope of a zero-intercept linear fit of three different time points from independent reactions. Error bars correspond to 1-σ (68.3%) confidence intervals for the slope.

FIG. 6 illustrates the cyclopropanation activities of variant 9-10A-TS-F87V-T268A (also called BM3-CIS or P450_(BM3)-CIS) and BM3-CIS-C400S (also called ABC-CIS or P411_(BM3)-CIS) driven by sodium dithionite under anaerobic and aerobic conditions. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value.

FIG. 7 illustrates the cyclopropanation and epoxidation activities of variant 9-10A-TS-F87V-T268A (also called BM3-CIS or P450_(BM3)-CIS) and BM3-CIS-C400S (also called ABC-CIS or P411_(BM3-CIS)) driven by NADPH under anaerobic and aerobic conditions. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Reaction conditions: 20 μM catalyst, 30 mM styrene, 10 mM EDA, 0.5 mM NADPH, 25 mM glucose, 2 U ml⁻¹ glucose dehydrogenase under argon (or air) in aqueous potassium phosphate buffer (pH 8.0) and 5% MeOH cosolvent for 2 hours at 298 K. Measurements were taken in triplicate and the error bars represent the standard deviation of the measurements.

FIGS. 8A-B illustrate the initial velocities plot for BM3-CIS-C400S (also called ABC-CIS or P411BM3-CIS)_(heme). (FIG. 8A) EDA concentration was varied at a saturating concentration of styrene (30 mM). (FIG. 8B) Styrene concentration was varied at a fixed concentration of EDA (20 mM). Initial rates were computed as the slope of a zero-intercept linear fit of three different time points from independent reactions. Error bars correspond to 1-σ (68.3%) confidence intervals for the slope.

FIG. 9 illustrates the heme domain active site and protein alignments of variant 9-10A-TS-F87V-T268A (also called BM3-CIS or P450_(BM3)-CIS) with BM3-CIS-C400S (also called ABC-CIS or P411_(BM3)-CIS) and wild type P450_(BM3). Top panels shows alignments of P450_(BM3)-CIS (green) and P411_(BM3)-CIS (peach) with left, middle and right panels showing active site residues, the active site I-helix, and global protein fold, respectively. No significant structural changes were observed (RMSD 0.52 Å). Middle panels: Large variations are observed upon comparing P411_(BM3)-CIS with the open (ligand-free) form of wild type P450_(BM3) (purple, taken from PDB#21J2, RMSD 1.2 Å). Pronounced rearrangements are observed in active site side chain residues (left) as well as rotations within the I-helix. Global movements are also observed in the N-terminal beta domain as well as F- and G-helices (right, marked by double headed arrows). These movements are consistent with well-known transitions that occur upon substrate binding and are important for native monooxygenation catalysis. Bottom panels: Alignment of P450_(BM3)-CIS with a ligand-bound P450_(BM3) structure (cyan, taken from PDB#1JPZ, RMSD 0.52 Å) demonstrates that P450_(BM3)-CIS and P411_(BM3)-CIS mimic the closed protein conformation even in the absence of substrate. Protein alignments were carried out using the align tool of PyMol (PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC.).

FIG. 10 illustrates that whole-cell cyclopropanation catalysts are inhibited under aerobic conditions. Variant 9-10A-TS-F87V-T268A=BM3-CIS and ABC=BM3+C400S. Reaction conditions: 20 mM styrene, 10 mM EDA, under argon (or air) in nitrogen-free medium and 5% MeOH cosolvent for 2 hours at 298 K (OD₆₀₀˜24). Total turnover=concentration of cyclopropanes (mM)/cell density (g cdw/L) in units of mmol/g cdw.

FIG. 11 illustrates the effect of glucose addition on in vivo cyclopropanation of styrene. Reaction conditions: 20 mM styrene, 10 mM EDA, under argon in nitrogen-free medium and 5% MeOH cosolvent for 2 hours at 298 K (OD₆₀₀˜24). Total turnover=concentration of cyclopropanes (mM)/cell density (g cdw/L) in units of mmol/g cdw. Variant 9-10A-TS-F87V-T268A=BM3-CIS and ABC=BM3+C400S.

FIG. 12 illustrates the effect of media and comparison of holo vs. heme forms of BM3-CIS-C400S on in vivo cyclopropanation of styrene. Reaction conditions: 20 mM styrene, 10 mM EDA, 2 mM glucose under argon in nitrogen-free medium and 5% MeOH cosolvent for 2 hours at 298 K (OD₆₀₀˜24). Variant 9-10A-TS-F87V-T268A=BM3-CIS and ABC=BM3+C400S.

FIG. 13 illustrates that increasing ABC catalyst loading (cell density) increases cyclopropanes yield up to approximately 80% at OD₆₀₀˜50. ABC (BM3-C400S or P411_(BM3)).

FIG. 14 illustrates the effect of using 1, 2, 3, 4 and 5 equivalents of styrene on reaction yield. Excess styrene gives only small improvements in yield. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. ABC (BM3-C400S or P411_(BM3)).

FIG. 15 illustrates controls for ABC catalyzed cyclopropanation. Variant 9-10A-TS-F87V-T268A=BM3-CIS and ABC=BM3-C400S.

FIG. 16 illustrates that ABC catalyst is active for 3 hours. At OD₆₀₀=25, the P450 concentration in 0.85 μM, such that TTN>8,000. Variant 9-10A-TS-F87V-T268A=BM3-CIS and ABC=BM3-C400S.

FIGS. 17A-D illustrate the proposed substrate scope of P450-catalyzed cyclopropanation. (FIG. 17A) Diazo-compounds that can be used as the carbenoid precursor. (FIG. 17B) Olefin partners for reaction. (FIG. 17C) Cyclopropanation of geranyl acetone for synthesis of anthroplalone and noranthroplone. (FIG. 17D) Cyclopropanation of 2,5-dimethylhexa-2,4-diene in the synthesis of pyrethroid insecticides.

FIG. 18 illustrates UV-Vis absorption spectra of purified BM3-CIS_(heme) (3 μM, red line) and BM3-CIS-C400S_(heme) (2 μM, green line). Insert shows solutions of both proteins at approximately 1 mM.

FIGS. 19A-C illustrate (FIG. 19A) Cytochrome P450s inefficiently catalyze cyclopropanation using NAD(P)H as a reductant because the Fe^(III)/Fe^(II) redox potential for the low-spin resting state (E^(o)′_(Fe-cys)=−430 mV) is lower than that of NAD(P)⁺/NAD(P)H (E^(o)′₁=−320 mV). Replacement of the heme-cysteine ligand (Fe-Cys) with serine (Fe-Ser) increased the resting state reduction potential and allowed reduction by NAD(P)H to the active Fe^(II) species in vivo. (FIG. 19B) Close-up of the ABC-CIS active site (PDB: 4H24) superimposed with an F_(o)-F_(c) simulated annealing omit map contoured at 3 σ showing electron density (green mesh) corresponding to the bound heme and C400S mutation. Interconnected density between C400S and the heme iron is consistent with proximal heme ligation by the C400S side chain hydroxyl. The heme, C400S and additional active site amino acid side chains are shown as sticks. (FIG. 19C) Potentiometric redox titrations for BM3 (green circles) and ABC (blue triangles) with overlaid one-electron Nernst curves. Insets show spectral changes between ferric (dashed line) and ferrous (solid line) states. The changes in absorbance near 450-470 nm (BM3) and 420-440 (ABC) were used to determine the Fe^(II)/Fe^(III) ratio after reduction with dithionite. The reduction is reversible, and reoxidation by potassium ferricyanide shows little or no hysteresis. The midpoint potential of the serine-ligated mutant (−293 mV) is shifted 127 mV positive compared to WT (−420 mV).

FIGS. 20A-B illustrate the heme electron density of BM3-CIS-C400S (also called ABC-CIS or P411_(BM3)-CIS). Maximum likelihood weighted electron density maps of serine-ligated heme in P411_(BM3)-CIS. (FIG. 20A) Stereo image of heme bound to P411_(BM3)-CIS viewed from the top of the heme in the active site. (FIG. 20B) Stereo image of heme bound to P411_(BM3)-CIS rotated ˜90° from panel A shows clear indication of heme-iron ligation by the side chain hydroxyl of C400S. All atoms are shown as sticks. All electron density maps were contoured at σ=1.0. For perspective, in panel B, the main chain atoms of residues 399 and 401 are also shown as sticks.

FIG. 21 illustrates the absolute spectra for ferric (blue), dithionite reduced ferrous (red) and carbon monoxide bound ferrous (green) ABC-CIS_(heme). Soret bands (nm): Fe^(III), 404; Fe^(II), 410, 425; Fe^(II)-CO, 411. Fe^(II)-CO displays α and β bands at 532 and 565 nm. Insert shows the carbon monoxide ferrous (pink) and the dithionite reduced ferrous (yellow) enzymes at 4.5 μM protein concentration.

FIG. 22 illustrates the absolute spectra for ferric (blue), dithionite reduced ferrous (red) and carbon monoxide bound ferrous (green) ABC-CIS_(holo). Soret bands (nm): Fe^(III), 404; Fe^(II), 410, 422; Fe^(II)-CO, 411. Fe^(II)-CO displays α and β bands at 533 and 566 nm. Ferric spectrum displays a broad peak at 465 nm.

FIGS. 23A-B illustrate the difference spectra for ferrous carbonyl with respect to ferrous for: (FIG. 23A) ABC-CIS_(heme) and (FIG. 23B) ABC-CIS_(holo).

FIG. 24 illustrates the potentiometric redox titration for P450_(BM3-heme) with overlaid Nernst curve fit to E^(o)′=˜420 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

FIG. 25 illustrates the potentiometric redox titration for P411_(BM3-heme) with overlaid Nernst curve fit to E^(o)′=−293 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

FIG. 26 illustrates the potentiometric redox titration for P450_(BM3-heme)-CIS with overlaid Nernst curve fit to E^(o)′=−360 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

FIG. 27 illustrates the potentiometric redox titration for P411_(BM3-heme)-CIS with overlaid Nernst curve fit to E^(o)′=−265 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

FIG. 28 illustrates the cyclopropanation activity under anaerobic vs. aerobic conditions with dithionite in variant 9-10A-TS-F87V-T268A (also called BM3-CIS or P450_(BM3-heme)-CIS) and BM3-CIS-C400S (also called ABC-CIS or P411_(BM3-heme)-CIS). Measurements were taken in triplicate and the error bars represent the standard deviation of the measurements.

FIG. 29 illustrates the effect of adding exogenous glucose (2 mM) on olefin cyclopropanation catalyzed by E. coli whole cells expressing 9-10A-TS-F87V-T268A (also called BM3-CIS and P450_(BM3)-CIS) or BM3-CIS-C400S (also called ABC-CIS and P411_(BM3)-CIS).

FIG. 30 illustrates the thermostabilities of heme domains of BM3-CIS (P450_(BM3)-CIS in blue) and ABC-CIS (P411_(BM3)-CIS in red). The C400S mutation stabilizes the heme domain by +1.7° C. The T₅₀ is the temperature at which half of the enzyme population has unfolded. Error bars correspond to 1-6 (68.3%) confidence intervals for the T₅₀.

FIG. 31 illustrates the effect of dioxygen exposure on whole-cell catalyzed cyclopropanation. ABC-CIS (P411_(BM3)-CIS) is strongly inhibited by dioxygen in vivo. All reactions had a cell density equivalent to OD₆₀₀=25. Reactions were conducted in the absence of exogenous glucose. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Product formation is defined as the amount of cyclopropane product (mmol) formed per mass of catalyst (g_(cdw)).

FIG. 32 illustrates the empty plasmid, no-induction controls and dithionite addition to whole cells. E. coli cells carrying the ABC-CIS (P411_(BM3)-CIS) gene but grown without the addition of IPTG (ABC-CIS no induction); E. coli cells carrying the pcWori plasmid but not the ABC-CIS gene (empty pcWori); ABC-CIS reaction with the addition of exogenous dithionite instead of glucose (ABC-CIS+dithionite). Reactions were left for two hours at 298 K. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Product formation is defined as the amount of cyclopropane product (mmol) formed per mass of catalyst (g_(cdw)).

FIG. 33 illustrates that increasing cell density increases cyclopropane yields up ˜80%. Total turnovers do not increase for cell densities higher than OD₆₀₀=20. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Product formation is defined as the amount of cyclopropane product (mmol) formed per mass of catalyst (g_(cdw)).

FIG. 34 illustrates the time-course for in vivo and in vitro ABC-CIS (P411_(BM3)-CIS)-catalyzed reaction at P411 loading of 1.6 μM [ε₄₁₁₋₄₉₀=103 cm⁻¹ for the ferrous-CO complex (K. P. Vatsis et al., J. Inorg. Biochem. 91, 542 (2002))]. Reaction conditions were as follows: 40 mM styrene, 20 mM EDA, 2 mM glucose, 10.2 g_(cdw), L⁻¹ whole-cell ABC-CIS (in vivo), 1.6 μM purified ABC-CIS (in vitro) in aqueous nitrogen-free M9 minimal medium and 5% MeOH cosolvent under anaerobic conditions at 298 K. Yields at each time point are reported as averages of two independent reactions.

FIGS. 35A-C illustrate the contrasting P450- and P411-mediated cyclopropanation. (FIG. 35A) Cytochrome P450s inefficiently catalyze cyclopropanation using NAD(P)H as a reductant because the Fe^(III)/Fe^(II) redox potential for the low-spin resting state (E^(o′) _(Fe-cys)=−430 mV) is lower than that of NAD(P)⁺/NAD(P)H (E^(o)′₁=−320 mV, right). Mutation of the heme-ligating Cys to Ser allows NAD(P)H-driven cyclopropanation while removing native monooxygenation (left). (FIG. 35B) Close-up of the P411_(BM3-heme)-CIS active site (PDB: 4H24) superimposed with an F_(o)-F_(c) simulated annealing omit map contoured at 3 σ showing electron density (green mesh) corresponding to the bound heme and C400S mutation. Heme, C400S and additional active site amino acid side chains are shown as sticks. (FIG. 35C) In vitro cyclopropanation vs. epoxidation of styrene catalyzed by P450_(BM3)-CIS and P411_(BM3)-CIS under anaerobic and aerobic conditions. Reaction conditions were as follows: 30 mM styrene, 10 mM EDA, 0.5 mM NADPH, 25 mM glucose, 2 U ml⁻¹ glucose dehydrogenase and 20 μg enzyme in aqueous potassium phosphate buffer and 5% MeOH cosolvent for six hours at 25° C. Error bars represent the standard deviation of three independent measurements.

FIGS. 36A-C illustrate the racemic synthesis of milnacipran. (FIG. 36A) Synthesis of key intermediate 1. (FIG. 36B) Routes A-C for conversion of 1 to milnacipran. (FIG. 36C) Route to milnacipran based on selective deprotonation of 5.

FIGS. 37A-B illustrate the enantioselective synthesis of levomilnacipran using (FIG. 37A) Rh-catalyzed intramolecular cyclopropanation or (FIG. 37B) asymmetric alkylation using tetraamine chiral auxiliary.

FIG. 38 illustrates the synthesis of levomilnacipran via carbene cyclopropanation by cytochrome P450_(BM3) variants.

FIG. 39 illustrates the gas chromatography trace of reaction of 9b and EDA mediated by BM3-CIS-AxH, using Agilent cycloSil-B column, 30 m×0.25 um×0.32 mm, method: 90° C. (hold 2 min), 90-110 (6° C./min), 110-190 (40° C./min), and 190-280 (20° C./min). Internal standard at 4.8 min, starting material at 10.4 min, and product at 14.8 min (E-isomer) and 15.4 min (Z-isomer).

FIG. 40 illustrates the proposed synthesis of bicifadine, DOV-216,303 and derivatives using P450-catalyzed cyclopropanation.

FIG. 41 illustrates that resting E. coli cells (425 μL) were purged with argon, before adding glucose (50 μL), 2,5-dimethyl-2,4-hexadiene (12.5 μL), and EDA (12.5 μL). The reaction was carried out in nitrogen free M9 minimal media with 5% methanol co-solvent for 24 hours at 298 K.

FIG. 42 illustrates a GC-FID chromatogram showing the two ethyl chrysanthemate diastereomers produced by BM3-T268A-C400S. Oven temperature: 90° C. for 2 min, then 2° C./min to 110° C., then 30° C./min to 230° C. HP-5 column (Agilent) 30 m×0.32 mm×0.25 μm.

FIG. 43 illustrates a GC-MS ion chromatogram (m=123) showing the two chrysanthemate diastereomers produced by BM3-T268A-C400S. Top insert shows the fragmentation pattern for ethyl chrysanthemate that gives rise to the molecular ion with m=123. Oven temperature: 90° C. for 2 min, then 2° C./min to 110° C., then 30° C./min to 230° C. HP-5 column (Agilent) 30 m×0.32 mm×0.25 μm.

FIG. 44 illustrates the chemical or enzymatic conversion of ethyl chrysanthemate to pyrethoid and pyrethrin insecticides. (a) Hydrolysis; (b) activation to the acid chloride; (c) coupling of the pyrethrolone alcohol (ROH) to the acid chloride; (d) transesterification.

FIG. 45A shows the sequence alignment between cytochrome P450 BM3 and CYP2D7. The C400 axial ligand in P450 BM3 corresponds to C461 in CYP2D6. FIG. 45B shows the sequence alignment between cytochrome P450 BM3 and P4502C7. The C400 axial ligand in P450 BM3 corresponds to C478 in P4502C7.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based on the surprising discovery that heme enzymes can be used to catalyze the conversion of olefins to any product containing one or more cyclopropane functional groups. In some aspects, cytochrome P450 enzymes (e.g., P450 BM3 (CYP102A1)) and variants thereof were identified as having an unexpectedly improved ability to catalyze the formal transfer of carbene equivalents from diazo reagents to various olefinic substrates, thereby making cyclopropane products with high regioselectivity and/or stereoselectivity. In particular embodiments, the present inventors have discovered that variants of P450_(BM3) with at least one or more amino acid mutations such as an axial ligand C400X (e.g., C400S) and/or an T268A amino acid substitution can catalyze cyclopropanation reactions efficiently, displaying increased total turnover numbers (TTN) and demonstrating highly regio- and enantioselective product formation compared to wild-type enzymes.

As a non-limiting example, axial serine heme ligation (C400S in BM3) in cytochrome P450s creates the homologous “cytochrome P411” family, which catalyze the cyclopropanation reaction in vivo in whole cells, providing over 10,000 total turnovers with high stereoselectivity, optical purity and yield, making the cyclopropane product with titers of over 20 g L⁻¹. Thus, the cytochrome P411 family is spectroscopically, electrochemically, and catalytically distinct from cytochrome P450s, providing a scaffold for engineering orthogonal heme-enzyme catalysis. As such, the ability to catalyze this non-natural C—C bond forming reaction in vivo advantageously expands the scope of transformations that are accessible to microbial organic synthesis and provides artificial metabolic pathways to complement nature's existing strategies for making cyclopropanes.

II. Definitions

The following definitions and abbreviations are to be used for the interpretation of the invention. The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment but encompasses all possible embodiments.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having”, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”

The term “cyclopropanation (enzyme) catalyst” or “enzyme with cyclopropanation activity” refers to any and all chemical processes catalyzed by enzymes, by which substrates containing at least one carbon-carbon double bond can be converted into cyclopropane products by using diazo reagents as carbene precursors.

The terms “engineered heme enzyme” and “heme enzyme variant” include any heme-containing enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing enzymes.

The terms “engineered cytochrome P450” and “cytochrome P450 variant” include any cytochrome P450 enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different cytochrome P450 enzymes.

The term “whole cell catalyst” includes microbial cells expressing heme-containing enzymes, wherein the whole cell catalyst displays cyclopropanation activity.

As used herein, the terms “porphyrin” and “metal-substituted porphyrins” include any porphyrin that can be bound by a heme enzyme or variant thereof. In particular embodiments, these porphyrins may contain metals including, but not limited to, Fe, Mn, Co, Cu, Rh, and Ru.

The terms “carbene equivalent” and “carbene precursor” include molecules that can be decomposed in the presence of metal (or enzyme) catalysts to structures that contain at least one divalent carbon with only 6 valence shell electrons and that can be transferred to C═C bonds to form cyclopropanes or to C—H or heteroatom-H bonds to form various carbon ligated products.

The terms “carbene transfer” and “foimal carbene transfer” as used herein include any chemical transformation where carbene equivalents are added to C═C bonds, carbon-heteroatom double bonds or inserted into C—H or heteroatom-H substrates.

As used herein, the terms “microbial,” “microbial organism” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.

As used herein, the term “non-naturally occurring”, when used in reference to a microbial organism or enzyme activity of the invention, is intended to mean that the microbial organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microbial organism or enzyme activity includes the cyclopropanation activity described above.

As used herein, the term “anaerobic”, when used in reference to a reaction, culture or growth condition, is intended to mean that the concentration of oxygen is less than about 25 μM, preferably less than about 5 μM, and even more preferably less than 1 μM. The term is also intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen. Preferably, anaerobic conditions are achieved by sparging a reaction mixture with an inert gas such as nitrogen or argon.

As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The term as it is used in reference to expression of an encoding nucleic acid refers to the introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.

The term “heterologous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.

On the other hand, the term “native” or “endogenous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.

The term “homolog,” as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. Thus, the term “homologous proteins” is intended to mean that the two proteins have similar amino acid sequences. In particular embodiments, the homology between two proteins is indicative of its shared ancestry, related by evolution.

The terms “analog” and “analogous” include nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.

As used herein, the term “alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term“alkenyl” refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl(ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “alkynyl” refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “aryl” refers to an aromatic carbon ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “cycloalkyl” refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, C₅₋₈, and C₆₋₈. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene. Cycloalkyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “heterocyclyl” refers to a saturated ring system having from 3 to 12 ring members and from 1 to 4 heteroatoms selected from N, O and S. Additional heteroatoms including, but not limited to, B, Al, Si and P can also be present in a heterocycloalkyl group. The heteroatoms can be oxidized to form moieties such as, but not limited to, —S(O)— and —S(O)₂—. Heterocyclyl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 4 to 6, or 4 to 7 ring members. Any suitable number of heteroatoms can be included in the heterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Examples of heterocyclyl groups include, but are not limited to, aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Heterocyclyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “heteroaryl” refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, O or S. Additional heteroatoms including, but not limited to, B, Al, Si and P can also be present in a heteroaryl group. The heteroatoms can be oxidized to form moieties such as, but not limited to, —S(O)— and —S(O)₂—. Heteroaryl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms can be included in the heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups can have from 5 to 8 ring members and from 1 to 4 heteroatoms, or from 5 to 8 ring members and from 1 to 3 heteroatoms, or from 5 to 6 ring members and from 1 to 4 heteroatoms, or from 5 to 6 ring members and from 1 to 3 heteroatoms. Examples of heteroaryl groups include, but are not limited to, pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Heteroaryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “alkoxy” refers to an alkyl group having an oxygen atom that connects the alkyl group to the point of attachment: i.e., alkyl-O—. As for alkyl group, alkoxy groups can have any suitable number of carbon atoms, such as C₁₋₆ or C₁₋₄. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the term “alkylthio” refers to an alkyl group having a sulfur atom that connects the alkyl group to the point of attachment: i.e., alkyl-S—. As for alkyl groups, alkylthio groups can have any suitable number of carbon atoms, such as C₁₋₆ or C₁₋₄. Alkylthio groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkylthio groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.

As used herein, the terms “halo” and “halogen” refer to fluorine, chlorine, bromine and iodine.

As used herein, the term “haloalkyl” refers to an alkyl moiety as defined above substituted with at least one halogen atom.

As used herein, the term “alkylsilyl” refers to a moiety —SiR₃, wherein at least one R group is alkyl and the other R groups are H or alkyl. The alkyl groups can be substituted with one more halogen atoms.

As used herein, the term “acyl” refers to a moiety —C(O)R, wherein R is an alkyl group.

As used herein, the term “oxo” refers to an oxygen atom that is double-bonded to a compound (i.e., O═).

As used herein, the term “carboxy” refers to a moiety —C(O)OH. The carboxy moiety can be ionized to form the carboxylate anion.

As used herein, the term “amino” refers to a moiety —NR₃, wherein each R group is H or alkyl.

As used herein, the term “amido” refers to a moiety —NRC(O)R or —C(O)NR₂, wherein each R group is H or alkyl.

III. DESCRIPTION OF THE EMBODIMENTS

In some aspects, the present invention provides methods for catalyzing the conversion of an olefin to any compound (e.g., any intermediate or final compound) containing one or more cyclopropane functional groups using heme enzymes. In certain aspects, the present invention provides a method for producing a cyclopropanation product, the method comprising:

-   -   (a) providing an olefinic substrate, a diazo reagent, and a heme         enzyme; and     -   (b) admixing the components of step (a) in a reaction for a time         sufficient to produce a cyclopropanation product.

In certain instances, the cyclopropanation product is produced via an intermolecular cyclopropanation reaction between the olefinic substrate and diazo reagent as separate distinct substrates. In other instances, the cyclopropanation product is produced via an intramolecular cyclopropanation reaction, e.g., wherein the olefinic substrate and diazo reagent are part of the same substrate.

The methods of the invention can be used to provide a vast number of cyclopropanation products. The cyclopropanation products include classes of compounds such as, but not limited to, insecticides (e.g., pyrethroid compounds), active pharmaceutical agents having chiral and/or achiral cyclopropane moieties (e.g., milnacipran, levomilnacipran, and other active ingredients such as antibiotics, antivirals, etc.), commodity and fine chemicals, plant hormones, flavors and scents, and fatty acids. The cyclopropanation products can also serve as intermediates for the synthesis of compounds belonging to these and other classes (e.g., chrysanthemate esters for the synthesis of pyrethroid compounds).

In other aspects, the present invention provides heme enzymes including variants and fragments thereof (e.g., truncated forms) as well as chimeric heme enzymes that are capable of carrying out the cyclopropanation reactions described herein. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention.

The following sections provide a description of exemplary and preferred embodiments including heme enzymes, expression vectors, host cells, cyclopropanation products such as, e.g., compounds comprising one or more cyclopropane functional groups, starting materials such as, e.g., olefinic substrates and diazo reagents, and characteristics and reaction conditions for the in vitro and in vivo cyclopropanation reactions described herein.

A. Heme Enzymes

In certain aspects, the present invention provides compositions comprising one or more heme enzymes that catalyze the conversion of an olefinic substrate to products containing one or more cyclopropane functional groups. In particular embodiments, the present invention provides heme enzyme variants comprising at least one or more amino acid mutations therein that catalyze the formal transfer of carbene equivalents from a diazo reagent (e.g., a diazo ester) to an olefinic substrate, making cyclopropane products with high stereoselectivity. In preferred embodiments, the heme enzyme variants of the present invention have the ability to catalyze cyclopropanation reactions efficiently, display increased total turnover numbers, and/or demonstrate highly regio- and/or enantioselective product formation compared to the corresponding wild-type enzymes.

The terms “heme enzyme” and “heme protein” are used herein to include any member of a group of proteins containing heme as a prosthetic group. Non-limiting examples of heme enzymes include globins, cytochromes, oxidoreductases, any other protein containing a heme as a prosthetic group, and combinations thereof. Heme-containing globins include, but are not limited to, hemoglobin, myoglobin, and combinations thereof. Heme-containing cytochromes include, but are not limited to, cytochrome P450, cytochrome b, cytochrome c1, cytochrome c, and combinations thereof. Heme-containing oxidoreductases include, but are not limited to, a catalase, an oxidase, an oxygenase, a haloperoxidase, a peroxidase, and combinations thereof.

In certain instances, the heme enzymes are metal-substituted heme enzymes containing protoporphyrin 1× or other porphyrin molecules containing metals other than iron, including, but not limited to, cobalt, rhodium, copper, ruthenium, and manganese, which are active cyclopropanation catalysts.

In some embodiments, the heme enzyme is a member of one of the enzyme classes set forth in Table 1. In other embodiments, the heme enzyme is a variant or homolog of a member of one of the enzyme classes set forth in Table 1. In yet other embodiments, the heme enzyme comprises or consists of the heme domain of a member of one of the enzyme classes set forth in Table 1 or a fragment thereof (e.g., a truncated heme domain) that is capable of carrying out the cyclopropanation reactions described herein.

TABLE 1 Heme enzymes identified by their enzyme classification number (EC number) and classification name. EC Number Name 1.1.2.3 L-lactate dehydrogenase 1.1.2.6 polyvinyl alcohol dehydrogenase (cytochrome) 1.1.2.7 methanol dehydrogenase (cytochrome c) 1.1.5.5 alcohol dehydrogenase (quinone) 1.1.5.6 formate dehydrogenase-N: 1.1.9.1 alcohol dehydrogenase (azurin): 1.1.99.3 gluconate 2-dehydrogenase (acceptor) 1.1.99.11 fructose 5-dehydrogenase 1.1.99.18 cellobiose dehydrogenase (acceptor) 1.1.99.20 alkan-1-ol dehydrogenase (acceptor) 1.2.1.70 glutamyl-tRNA reductase 1.2.3.7 indole-3-acetaldehyde oxidase 1.2.99.3 aldehyde dehydrogenase (pyrroloquinoline-quinone) 1.3.1.6 fumarate reductase (NADH): 1.3.5.1 succinate dehydrogenase (ubiquinone) 1.3.5.4 fumarate reductase (menaquinone) 1.3.99.1 succinate dehydrogenase 1.4.9.1 methylamine dehydrogenase (amicyanin) 1.4.9.2. aralkylamine dehydrogenase (azurin) 1.5.1.20 methylenetetrahydrofolate reductase [NAD(P)H] 1.5.99.6 spermidine dehydrogenase 1.6.3.1 NAD(P)H oxidase 1.7.1.1 nitrate reductase (NADH) 1.7.1.2 Nitrate reductase [NAD(P)H] 1.7.1.3 nitrate reductase (NADPH) 1.7.1.4 nitrite reductase [NAD(P)H] 1.7.1.14 nitric oxide reductase [NAD(P), nitrous oxide-forming] 1.7.2.1 nitrite reductase (NO-forming) 1.7.2.2 nitrite reductase (cytochrome; ammonia-forming) 1.7.2.3 trimethylamine-N-oxide reductase (cytochrome c) 1.7.2.5 nitric oxide reductase (cytochrome c) 1.7.2.6 hydroxylamine dehydrogenase 1.7.3.6 hydroxylamine oxidase (cytochrome) 1.7.5.1 nitrate reductase (quinone) 1.7.5.2 nitric oxide reductase (menaquinol) 1.7.6.1 nitrite dismutase 1.7.7.1 ferredoxin-nitrite reductase 1.7.7.2 ferredoxin-nitrate reductase 1.7.99.4 nitrate reductase 1.7.99.8 hydrazine oxidoreductase 1.8.1.2 sulfite reductase (NADPH) 1.8.2.1 sulfite dehydrogenase 1.8.2.2 thiosulfate dehydrogenase 1.8.2.3 sulfide-cytochrome-c reductase (flavocytochrome c) 1.8.2.4 dimethyl sulfide:cytochrome c2 reductase 1.8.3.1 sulfite oxidase 1.8.7.1 sulfite reductase (ferredoxin) 1.8.98.1 CoB-CoM heterodisulfide reductase 1.8.99.1 sulfite reductase 1.8.99.2 adenylyl-sulfate reductase 1.8.99.3 hydrogensulfite reductase 1.9.3.1 cytochrome-c oxidase 1.9.6.1 nitrate reductase (cytochrome) 1.10.2.2 ubiquinol-cytochrome-c reductase 1.10.3.1 catechol oxidase 1.10.3.B1 caldariellaquinol oxidase (H+-transporting) 1.10.3.3 L-ascorbate oxidase 1.10.3.9 photosystem II 1.10.3.10 ubiquinol oxidase (H+-transporting) 1.10.3.11 ubiquinol oxidase 1.10.3.12 menaquinol oxidase (H+-transporting) 1.10.9.1 plastoquinol-plastocyanin reductase 1.11.1.5 cytochrome-c peroxidase 1.11.1.6 catalase 1.11.1.7 peroxidase 1.11.1.B2 chloride peroxidase (vanadium-containing) 1.11.1.B7 bromide peroxidase (heme-containing) 1.11.1.8 iodide peroxidase 1.11.1.10 chloride peroxidase 1.11.1.11 L-ascorbate peroxidase 1.11.1.13 manganese peroxidase 1.11.1.14 lignin peroxidase 1.11.1.16 versatile peroxidase 1.11.1.19 dye decolorizing peroxidase 1.11.1.21 catalase-peroxidase 1.11.2.1 unspecific peroxygenase 1.11.2.2 myeloperoxidase 1.11.2.3 plant seed peroxygenase 1.11.2.4 fatty-acid peroxygenase 1.12.2.1 cytochrome-c3 hydrogenase 1.12.5.1 hydrogen:quinone oxidoreductase 1.12.99.6 hydrogenase (acceptor) 1.13.11.9 2,5-dihydroxypyridine 5,6-dioxygenase 1.13.11.11 tryptophan 2,3-dioxygenase 1.13.11.49 chlorite O2-lyase 1.13.11.50 acetylacetone-cleaving enzyme 1.13.11.52 indoleamine 2,3-dioxygenase 1.13.11.60 linoleate 8R-lipoxygenase 1.13.99.3 tryptophan 2′-dioxygenase 1.14.11.9 flavanone 3-dioxygenase 1.14.12.17 nitric oxide dioxygenase 1.14.13.39 nitric-oxide synthase (NADPH dependent) 1.14.13.17 cholesterol 7alpha-monooxygenase 1.14.13.41 tyrosine N-monooxygenase 1.14.13.70 sterol 14alpha-demethylase 1.14.13.71 N-methylcoclaurine 3′-monooxygenase 1.14.13.81 magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase 1.14.13.86 2-hydroxyisoflavanone synthase 1.14.13.98 cholesterol 24-hydroxylase 1.14.13.119 5-epiaristolochene 1,3-dihydroxylase 1.14.13.126 vitamin D3 24-hydroxylase 1.14.13.129 beta-carotene 3-hydroxylase 1.14.13.141 cholest-4-en-3-one 26-monooxygenase 1.14.13.142 3-ketosteroid 9alpha-monooxygenase 1.14.13.151 linalool 8-monooxygenase 1.14.13.156 1,8-cineole 2-endo-monooxygenase 1.14.13.159 vitamin D 25-hydroxylase 1.14.14.1 unspecific monooxygenase 1.14.15.1 camphor 5-monooxygenase 1.14.15.6 cholesterol monooxygenase (side-chain-cleaving) 1.14.15.8 steroid 15beta-monooxygenase 1.14.15.9 spheroidene monooxygenase 1.14.18.1 tyrosinase 1.14.19.1 stearoyl-CoA 9-desaturase 1.14.19.3 linoleoyl-CoA desaturase 1.14.21.7 biflaviolin synthase 1.14.99.1 prostaglandin-endoperoxide synthase 1.14.99.3 heme oxygenase 1.14.99.9 steroid 17alpha-monooxygenase 1.14.99.10 steroid 21-monooxygenase 1.14.99.15 4-methoxybenzoate monooxygenase (O-demethylating) 1.14.99.45 carotene epsilon-monooxygenase 1.16.5.1 ascorbate ferrireductase (transmembrane) 1.16.9.1 iron:rusticyanin reductase 1.17.1.4 xanthine dehydrogenase 1.17.2.2 lupanine 17-hydroxylase (cytochrome c) 1.17.99.1 4-methylphenol dehydrogenase (hydroxylating) 1.17.99.2 ethylbenzene hydroxylase 1.97.1.1 chlorate reductase 1.97.1.9 selenate reductase 2.7.7.65 diguanylate cyclase 2.7.13.3 histidine kinase 3.1.4.52 cyclic-guanylate-specific phosphodiesterase 4.2.1.B9 colneleic acid/etheroleic acid synthase 4.2.1.22 Cystathionine beta-synthase 4.2.1.92 hydroperoxide dehydratase 4.2.1.212 colneleate synthase 4.3.1.26 chromopyrrolate synthase 4.6.1.2 guanylate cyclase 4.99.1.3 sirohydrochlorin cobaltochelatase 4.99.1.5 aliphatic aldoxime dehydratase 4.99.1.7 phenylacetaldoxime dehydratase 5.3.99.3 prostaglandin-E synthase 5.3.99.4 prostaglandin-I synthase 5.3.99.5 Thromboxane-A synthase 5.4.4.5 9,12-octadecadienoate 8-hydroperoxide 8R-isomerase 5.4.4.6 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase 6.6.1.2 cobaltochelatase

In particular embodiments, the heme enzyme is a variant or a fragment thereof (e.g., a truncated variant containing the heme domain) comprising at least one mutation such as, e.g., a mutation at the axial position of the heme coordination site. In some instances, the mutation is a substitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the axial position. In certain instances, the mutation is a substitution of Cys with any other amino acid such as Ser at the axial position.

In certain embodiments, the in vitro methods for producing a cyclopropanation product comprise providing a heme enzyme, variant, or homolog thereof with a reducing agent such as NADPH or a dithionite salt (e.g., Na₂S₂O₄). In certain other embodiments, the in vivo methods for producing a cyclopropanation product comprise providing whole cells such as E. coli cells expressing a heme enzyme, variant, or homolog thereof.

In some embodiments, the heme enzyme, variant, or homolog thereof is recombinantly expressed and optionally isolated and/or purified for carrying out the in vitro cyclopropanation reactions of the present invention. In other embodiments, the heme enzyme, variant, or homolog thereof is expressed in whole cells such as E. coli cells, and these cells are used for carrying out the in vivo cyclopropanation reactions of the present invention.

In certain embodiments, the heme enzyme, variant, or homolog thereof comprises or consists of the same number of amino acid residues as the wild-type enzyme (e.g., a full-length polypeptide). In some instances, the heme enzyme, variant, or homolog thereof comprises or consists of an amino acid sequence without the start methionine (e.g., P450 BM3 amino acid sequence set forth in SEQ ID NO:1). In other embodiments, the heme enzyme comprises or consists of a heme domain fused to a reductase domain. In yet other embodiments, the heme enzyme does not contain a reductase domain, e.g., the heme enzyme contains a heme domain only or a fragment thereof such as a truncated heme domain.

In some embodiments, the heme enzyme, variant, or homolog thereof has an enhanced cyclopropanation activity of at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold compared to the corresponding wild-type heme enzyme.

In some embodiments, the heme enzyme, variant, or homolog thereof has a resting state reduction potential higher than that of NADH or NADPH.

In particular embodiments, the heme enzyme comprises a cyclochrome P450 enzyme. Cytochrome P450 enzymes constitute a large superfamily of heme-thiolate proteins involved in the metabolism of a wide variety of both exogenous and endogenous compounds. Usually, they act as the terminal oxidase in multicomponent electron transfer chains, such as P450-containing monooxygenase systems. Members of the cytochrome P450 enzyme family catalyze myriad oxidative transformations, including, e.g., hydroxylation, epoxidation, oxidative ring coupling, heteratom release, and heteroatom oxygenation (E. M. Isin et al., Biochim. Biophys. Acta 1770, 314 (2007)). The active site of these enzymes contains an Fe^(III)-protoporphyrin IX cofactor (heme) ligated proximally by a conserved cysteine thiolate (M. T. Green, Current Opinion in Chemical Biology 13, 84 (2009)). The remaining axial iron coordination site is occupied by a water molecule in the resting enzyme, but during native catalysis, this site is capable of binding molecular oxygen. In the presence of an electron source, typically provided by NADH or NADPH from an adjacent fused reductase domain or an accessory cytochrome P450 reductase enzyme, the heme center of cytochrome P450 activates molecular oxygen, generating a high valent iron(IV)-oxo porphyrin cation radical species intermediate (Compound I, FIG. 1) and a molecule of water.

One skilled in the art will appreciate that the cytochrome P450 enzyme superfamily has been compiled in various databases, including, but not limited to, the P450 homepage (available at http://dmelson.uthsc.edu/CytochromeP450.html; see also, D. R. Nelson, Hum. Genomics 4, 59 (2009)), the cytochrome P450 enzyme engineering database (available at http://www.cyped.uni-stuttgart.de/cgi-bin/CYPED5/index.pl; see also, D. Sirim et al., BMC Biochem 10, 27 (2009)), and the SuperCyp database (available at http://bioinformatics.charite.de/supercyp/; see also, S. Preissner et al., Nucleic Acids Res. 38, D237 (2010)), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

In certain embodiments, the cytochrome P450 enzymes of the invention are members of one of the classes shown in Table 2 (see, http://www.icgeb.org/˜p450srv/P450enzymes.html, the disclosure of which is incorporated herein by reference in its entirety for all purposes).

TABLE 2 Cytochrome P450 enzymes classified by their EC number, recommended name, and family/gene name. EC Recommended name Family/gene 1.3.3.9 secologanin synthase CYP72A1 1.14.13.11 trans-cinnamate 4-monooxygenase CYP73 1.14.13.12 benzoate 4-monooxygenase CYP53 1.14.13.13 calcidiol 1-monooxygenase CYP27 1.14.13.15 cholestanetriol 26-monooxygenase CYP27 1.14.13.17 cholesterol 7α-monooxygenase CYP7 1.14.13.21 flavonoid 3′-monooxygenase CYP75 1.14.13.28 3,9-dihydroxypterocarpan 6a-monooxygenase CYP93A1 1.14.13.30 leukotriene-B₄ 20-monooxygenase CYP4F 1.14.13.37 methyltetrahydroprotoberberine CYP93A1 14-monooxygenase 1.14.13.41 tyrosine N-monooxygenase CYP79 1.14.13.42 hydroxyphenylacetonitrile 2-monooxygenase — 1.14.13.47 (−)-limonene 3-monooxygenase — 1.14.13.48 (−)-limonene 6-monooxygenase — 1.14.13.49 (−)-limonene 7-monooxygenase — 1.14.13.52 isoflavone 3′-hydroxylase — 1.14.13.53 isoflavone 2′-hydroxylase — 1.14.13.55 protopine 6-monooxygenase — 1.14.13.56 dihydrosanguinarine 10-monooxygenase — 1.14.13.57 dihydrochelirubine 12-monooxygenase — 1.14.13.60 27-hydroxycholesterol 7α-monooxygenase — 1.14.13.70 sterol 14-demethylase CYP51 1.14.13.71 N-methylcoclaurine 3′-monooxygenase CYP80B1 1.14.13.73 tabersonine 16-hydroxylase CYP71D12 1.14.13.74 7-deoxyloganin 7-hydroxylase — 1.14.13.75 vinorine hydroxylase — 1.14.13.76 taxane 10β-hydroxylase CYP725A1 1.14.13.77 taxane 13α-hydroxylase CYP725A2 1.14.13.78 ent-kaurene oxidase CYP701 1.14.13.79 ent-kaurenoic acid oxidase CYP88A 1.14.14.1 unspecific monooxygenase multiple 1.14.15.1 camphor 5-monooxygenase CYP101 1.14.15.3 alkane 1-monooxygenase CYP4A 1.14.15.4 steroid 1β-monooxygenase CYP11B 1.14.15.5 corticosterone 18-monooxygenase CYP11B 1.14.15.6 cholesterol monooxygenase (side-chain- CYP11A cleaving) 1.14.21.1 (S)-stylopine synthase — 1.14.21.2 (S)-cheilanthifoline synthase — 1.14.21.3 berbamunine synthase CYP80 1.14.21.4 salutaridine synthase — 1.14.21.5 (S)-canadine synthase — 1.14.99.9 steroid 17α-monooxygenase CYP17 1.14.99.10 steroid 21-monooxygenase CYP21 1.14.99.22 ecdysone 20-monooxygenase — 1.14.99.28 linalool 8-monooxygenase CYP111 4.2.1.92 hydroperoxide dehydratase CYP74 5.3.99.4 prostaglandin-I synthase CYP8 5.3.99.5 thromboxane-A synthase CYP5

Table 3 below lists additional cyclochrome P450 enzymes that are suitable for use in the cyclopropanation reactions of the present invention. The accession numbers in Table 3 are incorporated herein by reference in their entirety for all purposes. The cytochrome P450 gene and/or protein sequences disclosed in the following patent documents are hereby incorporated by reference in their entirety for all purposes: WO 2013/076258; CN 103160521; CN 103223219; KR 2013081394; JP 5222410; WO 2013/073775; WO 2013/054890; WO 2013/048898; WO 2013/031975; WO 2013/064411; U.S. Pat. No. 8,361,769; WO 2012/150326, CN 102747053; CN 102747052; JP 2012170409; WO 2013/115484; CN 103223219; KR 2013081394; CN 103194461; JP 5222410; WO 2013/086499; WO 2013/076258; WO 2013/073775; WO 2013/064411; WO 2013/054890; WO 2013/031975; U.S. Pat. No. 8,361,769; WO 2012/156976; WO 2012/150326; CN 102747053; CN 102747052; US 20120258938; JP 2012170409; CN 102399796; JP 2012055274; WO 2012/029914; WO 2012/028709; WO 2011/154523; JP 2011234631; WO 2011/121456; EP 2366782; WO 2011/105241; CN 102154234; WO 2011/093185; WO 2011/093187; WO 2011/093186; DE 102010000168; CN 102115757; CN 102093984; CN 102080069; JP 2011103864; WO 2011/042143; WO 2011/038313; JP 2011055721; WO 2011/025203; JP 2011024534; WO 2011/008231; WO 2011/008232; WO 2011/005786; IN 2009DE01216; DE 102009025996; WO 2010/134096; JP 2010233523; JP 2010220609; WO 2010/095721; WO 2010/064764; US 20100136595; JP 2010051174; WO 2010/024437; WO 2010/011882; WO 2009/108388; US 20090209010; US 20090124515; WO 2009/041470; KR 2009028942; WO 2009/039487; WO 2009/020231; JP 2009005687; CN 101333520; CN 101333521; US 20080248545; JP 2008237110; CN 101275141; WO 2008/118545; WO 2008/115844; CN 101255408; CN 101250506; CN 101250505; WO 2008/098198; WO 2008/096695; WO 2008/071673; WO 2008/073498; WO 2008/065370; WO 2008/067070; JP 2008127301; JP 2008054644; KR 794395; EP 1881066; WO 2007/147827; CN 101078014; JP 2007300852; WO 2007/048235; WO 2007/044688; WO 2007/032540; CN 1900286; CN 1900285; JP 2006340611; WO 2006/126723; KR 2006029792; KR 2006029795; WO 2006/105082; WO 2006/076094; US 2006/0156430; WO 2006/065126; JP 2006129836; CN 1746293; WO 2006/029398; JP 2006034215; JP 2006034214; WO 2006/009334; WO 2005/111216; WO 2005/080572; US 2005/0150002; WO 2005/061699; WO 2005/052152; WO 2005/038033; WO 2005/038018; WO 2005/030944; JP 2005065618; WO 2005/017106; WO 2005/017105; US 20050037411; WO 2005/010166; JP 2005021106; JP 2005021104; JP 2005021105; WO 2004/113527; CN 1472323; JP 2004261121; WO 2004/013339; WO 2004/011648; DE 10234126; WO 2004/003190; WO 2003/087381; WO 2003/078577; US 20030170627; US 20030166176; US 20030150025; WO 2003/057830; WO 2003/052050; CN 1358756; US 20030092658; US 20030078404; US 20030066103; WO 2003/014341; US 20030022334; WO 2003/008563; EP 1270722; US 20020187538; WO 2002/092801; WO 2002/088341; US 20020160950; WO 2002/083868; US 20020142379; WO 2002/072758; WO 2002/064765; US 20020076777; US 20020076774; US 20020076774; WO 2002/046386; WO 2002/044213; US 20020061566; CN 1315335; WO 2002/034922; WO 2002/033057; WO 2002/029018; WO 2002/018558; JP 2002058490; US 20020022254; WO 2002/008269; WO 2001/098461; WO 2001/081585; WO 2001/051622; WO 2001/034780; CN 1271005; WO 2001/011071; WO 2001/007630; WO 2001/007574; WO 2000/078973; U.S. Pat. No. 6,130,077; JP 2000152788; WO 2000/031273; WO 2000/020566; WO 2000/000585; DE 19826821; JP 11235174; U.S. Pat. No. 5,939,318; WO 99/19493; WO 99/18224; U.S. Pat. No. 5,886,157; WO 99/08812; U.S. Pat. No. 5,869,283; JP 10262665; WO 98/40470; EP 776974; DE 19507546; GB 2294692; U.S. Pat. No. 5,516,674; JP 07147975; WO 94/29434; JP 06205685; JP 05292959; JP 04144680; DD 298820; EP 477961; SU 1693043; JP 01047375; EP 281245; JP 62104583; JP 63044888; JP 62236485; JP 62104582; and JP 62019084.

TABLE 3 Additional cytochrome P450 enzymes of the present invention. Species Cyp No. Accession No. SEQ ID NO Bacillus megaterium 102A1 AAA87602 1 Bacillus megaterium 102A1 ADA57069 2 Bacillus megaterium 102A1 ADA57068 3 Bacillus megaterium 102A1 ADA57062 4 Bacillus megaterium 102A1 ADA57061 5 Bacillus megaterium 102A1 ADA57059 6 Bacillus megaterium 102A1 ADA57058 7 Bacillus megaterium 102A1 ADA57055 8 Bacillus megaterium 102A1 ACZ37122 9 Bacillus megaterium 102A1 ADA57057 10 Bacillus megaterium 102A1 ADA57056 11 Mycobacterium sp. 153A6 CAH04396 12 HXN-1500 Tetrahymena thermophile 5013C2 ABY59989 13 Nonomuraea dietziae AGE14547.1 14 Homo sapiens 2R1 NP_078790 15 Macca mulatta 2R1 NP_001180887.1 16 Canis familiaris 2R1 XP_854533 17 Mus musculus 2R1 AAI08963 18 Bacillus halodurans C-125 152A6 NP_242623 19 Streptomyces parvus aryC AFM80022 20 Pseudomonas putida 101A1 P00183 21 Homo sapiens 2D7 AAO49806 22 Rattus norvegicus C27 AAB02287 23 Oryctolagus cuniculus 2B4 AAA65840 24 Bacillus subtilis 102A2 O08394 25 Bacillus subtilis 102A3 O08336 26 B. megaterium DSM 32 102A1 P14779 27 B. cereus ATCC14579 102A5 AAP10153 28 B. licheniformis 102A7 YP 079990 29 ATTC1458 B. thuringiensis X YP 037304 30 serovar konkukian str.97-27 R. metallidurans CH34 102E1 YP 585608 31 A. fumigatus Af293 505X EAL92660 32 A. nidulans FGSC A4 505A8 EAA58234 33 A. oryzae ATCC42149 505A3 Q2U4F1 34 A. oryzae ATCC42149 X Q2UNA2 35 F. oxysporum 505A1 Q9Y8G7 36 G. moniliformis X AAG27132 37 G. zeae PH1 505A7 EAA67736 38 G. zeae PH1 505C2 EAA77183 39 M. grisea 70-15 syn 505A5 XP 365223 40 N. crassa OR74 A 505A2 XP 961848 41 Oryza sativa* 97A Oryza sativa* 97B Oryza sativa 97C ABB47954 42 The start methionine (“M”) may be present or absent from these sequences. *See, M. Z. Lv et al., Plant Cell Physiol., 53(6): 987-1002 (2012).

In certain embodiments, the present invention provides amino acid substitutions that efficiently remove monooxygenation chemistry from cytochrome P450 enzymes. This system permits selective enzyme-driven cyclopropanation chemistry without competing side reactions mediated by native P450 catalysis. The invention also provides P450-mediated catalysis that is competent for cyclopropanation chemistry but not able to carry out traditional P450-mediated monooxygenation reactions as ‘orthogonal’ P450 catalysis and respective enzyme variants as ‘orthogonal’ P450s. In some instances, orthogonal P450 variants comprise a single amino acid mutation at the axial position of the heme coordination site (e.g., a C400S mutation in the P450 BM3 enzyme) that alters the proximal heme coordination environment. Accordingly, the present invention also provides P450 variants that contain an axial heme mutation in combination with one or more additional mutations described herein to provide orthogonal P450 variants that show enriched diastereoselective and/or enantioselective product distributions. The present invention further provides a compatible reducing agent for orthogonal P450 cyclopropanation catalysis that includes, but is not limited to, NAD(P)H or sodium dithionite.

In particular embodiments, the cytochrome P450 enzyme is one of the P450 enzymes or enzyme classes set forth in Table 2 or 3. In some embodiments, the cytochrome P450 enzyme is a variant or homolog of one of the P450 enzymes or enzyme classes set forth in Table 2 or 3. In preferred embodiments, the P450 enzyme variant comprises a mutation at the conserved cysteine (Cys or C) residue of the corresponding wild-type sequence that serves as the heme axial ligand to which the iron in protoporphyrin IX is attached. As non-limiting examples, axial mutants of any of the P450 enzymes set forth in Table 2 or 3 can comprise a mutation at the axial position (“A×X”) of the heme coordination site, wherein “X” is selected from Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

In certain embodiments, the conserved cysteine residue in a cytochrome P450 enzyme of interest that serves as the heme axial ligand and is attached to the iron in protoporphyrin IX can be identified by locating the segment of the DNA sequence in the corresponding cytochrome P450 gene which encodes the conserved cysteine residue. In some instances, this DNA segment is identified through detailed mutagenesis studies in a conserved region of the protein (see, e.g., Shimizu et al., Biochemistry 27, 4138-4141, 1988). In other instances, the conserved cysteine is identified through crystallographic study (see, e.g., Poulos et al., J. Mol. Biol. 195:687-700, 1987).

In situations where detailed mutagenesis studies and crystallographic data are not available for a cytochrome P450 enzyme of interest, the axial ligand may be identified through phylogenetic study. Due to the similarities in amino acid sequence between P450 enzymes, standard protein alignment algorithms may show a phylogenetic similarity between a P450 enzyme for which crystallographic or mutagenesis data exist and a new P450 enzyme for which such data do not exist. Thus, the polypeptide sequences of the present invention for which the heme axial ligand is known can be used as a “query sequence” to perform a search against a specifc new cytochrome P450 enzyme of interest or a database comprising cytochrome P450 sequences to identify the heme axial ligand. Such analyses can be performed using the BLAST programs (see, e.g., Altschul et al., J Mol Biol. 215(3):403-10 (1990)). Software for performing BLAST analyses publicly available through the National Center for Biotechnology Information (http://ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences.

Exemplary parameters for performing amino acid sequence alignments to identify the heme axial ligand in a P450 enzyme of interest using the BLASTP algorithm include E value=10, word size=3, Matrix=Blosum62, Gap opening=11, gap extension=1, and conditional compositional score matrix adjustment. Those skilled in the art will know what modifications can be made to the above parameters, e.g., to either increase or decrease the stringency of the comparison and/or to determine the relatedness of two or more sequences.

In preferred embodiments, the cytochrome P450 enzyme is a cytochrome P450 BM3 enzyme or a variant, homolog, or fragment thereof. The bacterial cytochrome P450 BM3 from Bacillus megaterium is a water soluble, long-chain fatty acid monooxygenase. The native P450 BM3 protein is comprised of a single polypeptide chain of 1048 amino acids and can be divided into 2 functional subdomains (see, L. O, Narhi et al., J. Biol. Chem. 261, 7160 (1986)). An N-terminal domain, amino acid residues 1-472, contains the heme-bound active site and is the location for monoxygenation catalysis. The remaining C-terminal amino acids encompass a reductase domain that provides the necessary electron equivalents from NADPH to reduce the heme cofactor and drive catalysis. The presence of a fused reductase domain in P450 BM3 creates a self-sufficient monooxygenase, obviating the need for exogenous accessory proteins for oxygen activation (see, id.). It has been shown that the N-terminal heme domain can be isolated as an individual, well-folded, soluble protein that retains activity in the presence of hydrogen peroxide as a terminal oxidant under appropriate conditions (P. C. Cirino et al., Angew. Chem., Int. Ed. 42, 3299 (2003)).

In certain instances, the cytochrome P450 BM3 enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO:1. In certain other instances, the cytochrome P450 BM3 enzyme is a natural variant thereof as described, e.g., in J. Y. Kang et al., AMB Express 1:1 (2011), wherein the natural variants are divergent in amino acid sequence from the wild-type cytochrome P450 BM3 enzyme sequence (SEQ ID NO:1) by up to about 5% (e.g., SEQ ID NOS:2-11).

In particular embodiments, the P450 BM3 enzyme variant comprises or consists of the heme domain of the wild-type P450 BM3 enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO:1) and optionally at least one mutation as described herein. In other embodiments, the P450 BM3 enzyme variant comprises or consists of a fragment of the heme domain of the wild-type P450 BM3 enzyme sequence (SEQ ID NO:1), wherein the fragment is capable of carrying out the cyclopropanation reactions of the present invention. In some instances, the fragment includes the heme axial ligand and at least one, two, three, four, or five of the active site residues.

In certain embodiments, the P450 BM3 enzyme variant comprises a mutation at the axial position (“A×X”) of the heme coordination site, wherein “X” is selected from Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. The conserved cysteine (Cys or C) residue in the wild-type P450 BM3 enzyme is located at position 400 in SEQ ID NO:1. As used herein, the terms “A×X” and “C400X” refer to the presence of an amino acid substitution “X” located at the axial position (i.e., residue 400) of the wild-type P450 BM3 enzyme (i.e., SEQ ID NO:1). In some instances, X is Ser (S). In other instances, X is Ala (A), Asp (D), His (H), Lys (K), Asn (N), Met (M), Thr (T), or Tyr (Y). In some embodiments, the P450 BM3 enzyme variant comprises or consists of the heme domain of the wild-type P450 BM3 enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO:1) or a fragment thereof and an A×X mutation (i.e., “WT-A×X heme”).

In other embodiments, the P450 BM3 enzyme variant comprises at least one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen) of the following amino acid substitutions in SEQ ID NO:1: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K. In certain instances, the P450 BM3 enzyme variant comprises a T268A mutation alone or in combination with one or more additional mutations such as a C400X mutation (e.g., C400S) in SEQ ID NO:1. In other instances, the P450 BM3 enzyme variant comprises all thirteen of these amino acid substitutions (i.e., V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, 1366V, and E442K; “BM3-CIS”) in combination with a C400X mutation (e.g., C400S) in SEQ ID NO:1. In some instances, the P450 BM3 enzyme variant comprises or consists of the heme domain of the BM3-CIS enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO:1 comprising all thirteen of these amino acid substitutions) or a fragment thereof and an “A×X” mutation (i.e., “BM3-CIS-A×X heme”).

In some embodiments, the P450 BM3 enzyme variant further comprises at least one or more (e.g., at least two, or all three) of the following amino acid substitutions in SEQ ID NO:1: I263A, A328G, and a T438 mutation. In certain instances, the T438 mutation is T438A, T438S, or T438P. In some instances, the P450 BM3 enzyme variant comprises a T438 mutation such as T438A, T438S, or T438P alone or in combination with one or more additional mutations such as a C400X mutation (e.g., C400S) in SEQ ID NO:1 or a heme domain or fragment thereof. In other instances, the P450 BM3 enzyme variant comprises a T438 mutation such as T438A, T438S, or T438P in a BM3-CIS backbone alone or in combination with a C400X mutation (e.g., C400S) in SEQ ID NO:1 (i.e., “BM3-CIS-T438S-A×X”). In yet other instances, the P450 BM3 enzyme variant comprises or consists of the heme domain of the BM3-CIS enzyme sequence or a fragment thereof in combination with a T438 mutation and an “A×X” mutation (e.g., “BM3-CIS-T438S-A×X heme”).

In other embodiments, the P450 BM3 enzyme variant further comprises from one to five (e.g., one, two, three, four, or five) active site alanine substitutions in the active site of SEQ ID NO:1. In certain instances, the active site alanine substitutions are selected from the group consisting of L75A, M177A, L181A, I263A, L437A, and a combination thereof.

In further embodiments, the P450 BM3 enzyme variant comprises at least one or more (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following amino acid substitutions in SEQ ID NO:1: R47C, L521, I58V, L75R, F81 (e.g., F81L, F81W), A82 (e.g., A82S, A82F, A82G, A82T, etc.), F87A, K94I, I94K, H100R, S106R, F107L, A135S, F162I, A197V, F205C, N239H, R255S, S274T, L324I, A328V, V340M, and K434E. In particular embodiments, the P450 BM3 enzyme variant comprises any one or a plurality of these mutations alone or in combination with one or more additional mutations such as those described above, e.g., an “A×X” mutation and/or at least one or more mutations including V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K.

Table 4 below provides non-limiting examples of cytochrome P450 BM3 variants of the present invention. Each P450 BM3 variant comprises one or more of the listed mutations (Variant Nos. 1-31), wherein a “+” indicates the presence of that particular mutation(s) in the variant. Any of the variants listed in Table 4 can further comprise an I263A and/or an A328G mutation and/or at least one, two, three, four, or five of the following alanine substitutions, in any combination, in the P450 BM3 enzyme active site: L75A, M177A, L181A, I263A, and L437A. In particular embodiments, the P450 BM3 variant comprises or consists of the heme domain of any one of Variant Nos. 1-31 listed in Table 4 or a fragment thereof, wherein the fragment is capable of carrying out the cyclopropanation reactions of the present invention.

TABLE 4 Exemplary cytochrome P450 BM3 enzyme variants of the present invention. P450_(BM3) variant Mutation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 C400X + + + + + + T268A + + + + + + F87V + + + + + + 9-10A-TS + + + + + T438Z + + + + + P450_(BM3) variant Mutation 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 C400X + + + + + + + + + + T268A + + + + + + + + + + F87V + + + + + + + + + + 9-10A-TS + + + + + + + + + + + T438Z + + + + + + + + + + + Mutations relative to the wild-type P450_(BM3) amino acid sequence (SEQ ID NO: 1); “X” is selected from Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; “Z” is selected from Ala, Ser, and Pro; “9-10A-TS” includes the following amino acid substitutions in SEQ ID NO: 1: V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, and E442K.

One skilled in the art will understand that any of the mutations listed in Table 4 can be introduced into any cytochrome P450 enzyme of interest by locating the segment of the DNA sequence in the corresponding cytochrome P450 gene which encodes the conserved amino acid residue as described above for identifying the conserved cysteine residue in a cytochrome P450 enzyme of interest that serves as the heme axial ligand. In certain instances, this DNA segment is identified through detailed mutagenesis studies in a conserved region of the protein (see, e.g., Shimizu et al., Biochemistry 27, 4138-4141, 1988). In other instances, the conserved amino acid residue is identified through crystallographic study (see, e.g., Poulos et al., J. Mol. Biol. 195:687-700, 1987). In yet other instances, protein sequence alignment algorithms can be used to identify the conserved amino acid residue. As non-limiting examples, the use of BLAST alignment with the P450 BM3 amino acid sequence as the query sequence to identify the heme axial ligand site and/or the equivalent T268 residue in other cytochrome P450 enzymes is illustrated in Examples 3 and 9.

Table 5A below provides non-limiting examples of preferred cytochrome P450 BM3 variants of the present invention. Table 5B below provides non-limiting examples of preferred chimeric cytochrome P450 enzymes of the present invention.

TABLE 5A Exemplary preferred cytochrome P450 BM3 enzyme variants of the invention. P450_(BM3) variants Mutations compared to wild-type P450_(BM3) (SEQ ID NO: 1) P450_(BM3)-T268A T268A P450_(BM3)-T268A-C400H T268A + C400H P411_(BM3) (ABC) C400S P411_(BM3)-T268A T268A + C400S P450_(BM3)-T268A-F87V T268A + F87V 9-10A TS V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, E442K 9-10A-TS-F87V 9-10A TS + F87V H2A10 9-10A TS + F87V, L75A, L181A, T268A H2A10-C400S 9-10A TS + F87V, L75A, L181A, T268A, C400S H2A10-C400H 9-10A TS + F87V, L75A, L181A, T268A, C400H H2A10-C400M 9-10A TS + F87V, L75A, L181A, T268A, C400M H2-5-F10 9-10A TS + F87V, L75A, I263A, T268A, L437A H2-5-F10-C400S 9-10A TS + F87V, L75A, I263A, T268A, L437A, C400S H2-5-F10-C400H 9-10A TS + F87V, L75A, I263A, T268A, L437A, C400H H2-5-F10-C400M 9-10A TS + F87V, L75A, I263A, T268A, L437A, C400M H2-4-D4 9-10A TS + F87V, L75A, M177A, L181A, T268A, L437A H2-4-D4-C400S 9-10A TS + F87V, L75A, M177A, L181A, T268A, L437A, C400S H2-4-D4-C400H 9-10A TS + F87V, L75A, M177A, L181A, T268A, L437A, C400H H2-2-A1 9-10A TS + F87A, L75A, L181A, L437A H2-2-A1-C400S 9-10A TS + F87A, L75A, L181A, L437A, C400S H2-2-A1-C400H 9-10A TS + F87A, L75A, L181A, L437A, C400H H2-8-C7 9-10A TS + L75A, F87V, L181A H2-5-F10-A75L 9-10A TS + F87V-I263A-T268A-L437A CH F8 R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, F81W, A82S, F87A, A197V BM3-CIS (P450_(BM3)-CIS; C3C) 9-10A TS + F87V, T268A BM3-CIS-I263A BM3-CIS + I263A BM3-CIS-A328G BM3-CIS + A328G BM3-CIS-T438S BM3-CIS + T438S BM3-CIS-C400S (P411_(BM3)-CIS; BM3-CIS + C400S ABC-CIS) BM3-CIS-C400D (BM3-CIS-AxD) BM3-CIS + C400D BM3-CIS-C400Y (BM3-CIS-AxY) BM3-CIS + C400Y BM3-CIS-C400K (BM3-CIS-AxK) BM3-CIS + C400K BM3-CIS-C400H (BM3-CIS-AxH) BM3-CIS + C400H BM3-CIS-C400M (BM3-CIS-AxM) BM3-CIS + C400M WT-AxA (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400A WT-AxD (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400D WT-AxH (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400H WT-AxK (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400K WT-AxM (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400M WT-AxN (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400N WT-AxY (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400Y BM3-CIS-T438S-AxA BM3-CIS-T438S + C400A BM3-CIS-T438S-AxD BM3-CIS-T438S + C400D BM3-CIS-T438S-AxM BM3-CIS-T438S + C400M BM3-CIS-T438S-AxY BM3-CIS-T438S + C400Y BM3-CIS-T438S-AxT BM3-CIS-T438S + C400T 7-11D R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, A82F, A328V 7-11D-C400S R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, A82F, A328V, C400S 12-10C R47C, V78A, A82G, F87V, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328V, L353V 22A3 L52I, I58V, L75R, F87A, H100R, S106R, F107L, A135S, F162I, A184V, N239H, S274T, L324I, V340M, I366V, K434E Man1 V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, F81L, A82T, F87A, I94K

TABLE 5B Exemplary preferred chimeric cytochrome P450 enzymes of the invention. Chimeric P450s Heme domain block sequence SEQ ID NO C2G9 22223132 43 X7 22312333 44 X7-12 12112333 45 C2E6 11113311 46 X7-9 32312333 47 C2B12 32313233 48 TSP234 22313333 49

In particular embodiments, cytochrome P450 BM3 variants with at least one or more amino acid mutations such as, e.g., C400X (A×X), BM3-CIS, T438, and/or T268A amino acid substitutions catalyze cyclopropanation reactions efficiently, displaying increased total turnover numbers and demonstrating highly regio- and/or enantioselective product formation compared to the wild-type enzyme.

As a non-limiting example, the cytochrome P450 BM3 variants of the present invention are cis-selective catalysts that demonstrate diastereomeric ratios at least comparable to wild-type P450 BM3, e.g., at least 37:63 cis:trans, at least 50:50 cis:trans, at least 60:40 cis:trans, or at least 95:5 cis:trans. Particular mutations for improving cis-selective catalysis include at least one mutation comprising T268A, C400X, and T438S, but preferably one, two, or all three of these mutations in combination with additional mutations comprising V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, E442K, and F87V derived from P450 BM3 variant 9-10A-TS. These mutations are isolated to the heme domain of P450 BM3 and are located in various regions of the heme domain structure including the active site and periphery.

As another non-limiting example, the cytochrome P450 BM3 variants of the present invention are trans-selective catalysts that demonstrate diastereomeric ratios at least comparable to wild-type P450 BM3, e.g., at least 37:63 cis:trans, at least 20:80 cis:trans, or at least 1:99 cis:trans. Particular mutations for improving trans-selective catalysis include at least one mutation comprising including T268A and C400X, but preferably one or both of these mutations in the background of wild-type P450 BM3. In certain embodiments, trans-preferential mutations in combination with additional mutations such as V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, E442K, and F87V (from 9-10-A-TS) are also tolerated when in the presence of additional mutations including, but not limited to, I263A, L437A, L181A and/or L75A. These mutations are isolated to the heme domain of P450 BM3 and are located in various regions of the heme domain structure including the active site and periphery.

In certain embodiments, the present invention also provides P450 variants that catalyze enantioselective cyclopropanation with enantiomeric excess values of at least 30% (comparable with wild-type P450 BM3), but more preferably at least 80%, and even more preferably at least >95% for preferred product diastereomers.

In other aspects, the present invention provides chimeric heme enzymes such as, e.g., chimeric P450 proteins comprised of recombined sequences from P450 BM3 and at least one, two, or more distantly related P450 enzymes from Bacillus subtillis or any other organism that are competent cyclopropanation catalysts using similar conditions to wild-type P450 BM3 and highly active P450 BM3 variants. As a non-limiting example, site-directed recombination of three bacterial cytochrome P450s can be performed with sequence crossover sites selected to minimize the number of disrupted contacts within the protein structure. In some embodiments, seven crossover sites can be chosen, resulting in eight sequence blocks. One skilled in the art will understand that the number of crossover sites can be chosen to produce the desired number of sequence blocks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 crossover sites for 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequence blocks, respectively. In other embodiments, the numbering used for the chimeric P450 refers to the identity of the parent sequence at each block. For example, “12312312” refers to a sequence containing block 1 from P450 #1, block 2 from P450 #2, block 3 from P450 #3, block 4 from P450 #1, block 5 from P450 #2, and so on. A chimeric library useful for generating the chimeric heme enzymes of the invention can be constructed as described in, e.g., Otey et al., PLoS Biology, 4(5):e112 (2006), following the SISDC method (see, Hiraga et al., J. Mol. Biol., 330:287-96 (2003)) using the type IIb restriction endonuclease BsaXI, ligating the full-length library into the pCWori vector and transforming into the catalase-deficient E. coli strain SN0037 (see, Nakagawa et al., Biosci. Biotechnol. Biochem., 60:415-420 (1996)); the disclosures of these references are hereby incorporated by reference in their entirety for all purposes.

As a non-limiting example, chimeric P450 proteins comprising recombined sequences or blocks of amino acids from CYP102A1 (Accession No. J04832), CYP102A2 (Accession No. CAB12544), and CYP102A3 (Accession No. U93874) can be constructed. In certain instances, the CYP102A1 parent sequence is assigned “1”, the CYP102A2 parent sequence is assigned “2”, and the CYP102A3 is parent sequence assigned “3”. In some instances, each parent sequence is divided into eight sequence blocks containing the following amino acids (aa): block 1: aa 1-64; block 2: aa 65-122; block 3: aa 123-166; block 4: aa 167-216; block 5: aa 217-268; block 6: aa 269-328; block 7: aa 329-404; and block 8: aa 405-end. Thus, in this example, there are eight blocks of amino acids and three fragments are possible at each block. For instance, “12312312” refers to a chimeric P450 protein of the invention containing block 1 (aa 1-64) from CYP102A1, block 2 (aa 65-122) from CYP102A2, block 3 (aa 123-166) from CYP102A3, block 4 (aa 167-216) from CYP102A1, block 5 (aa 217-268) from CYP102A2, and so on. See, e.g., Otey et al., PLoS Biology, 4(5):e112 (2006). Non-limiting examples of chimeric P450 proteins include those set forth in Table 5B (C2G9, X7, X7-12, C2E6, X7-9, C2B12, TSP234) and Table 13. In some embodiments, the chimeric heme enzymes of the invention can comprise at least one or more of the mutations described herein.

In some embodiments, the present invention provides the incorporation of homologous or analogous mutations to C400X (A×X) and/or T268A in other cytochrome P450 enzymes and heme enzymes in order to impart or enhance cyclopropanation activity.

As non-limiting examples, the cytochrome P450 can be a variant of CYP101A1 (SEQ ID NO:25) comprising a C357X (e.g., C357S) mutation, a T252A mutation, or a combination of C357X (e.g., C357S) and T252A mutations, wherein “X” is any amino acid other than Cys, or the cytochrome P450 can be a variant of CYP2B4 (SEQ ID NO:28) comprising a C436X (e.g., C436S) mutation, a T302A mutation, or a combination of C436X (e.g., C436S) and T302A mutations, wherein “X” is any amino acid other than Cys, or the cytochrome P450 can be a variant of CYP2D7 (SEQ ID NO:26) comprising a C461X (e.g., C461S) mutation, wherein “X” is any amino acid other than Cys, or the cytochrome P450 can be a variant of P450C27 (SEQ ID NO:27) comprising a C478X (e.g., C478S) mutation, wherein “X” is any amino acid other than Cys.

An enzyme's total turnover number (or TTN) refers to the maximum number of molecules of a substrate that the enzyme can convert before becoming inactivated. In general, the TTN for the heme enzymes of the invention range from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more. In certain embodiments, the variant or chimeric heme enzymes of the present invention have higher TTNs compared to the wild-type sequences. In some instances, the variant or chimeric heme enzymes have TTNs greater than about 100 (e.g., at least about 100, 150, 200, 250, 300, 325, 350, 400, 450, 500, or more) in carrying out in vitro cyclopropanation reactions. In other instances, the variant or chimeric heme enzymes have TTNs greater than about 1000 (e.g., at least about 1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000, 100,000, or more) in carrying out in vivo whole cell cyclopropanation reactions.

When whole cells expressing a heme enzyme are used to carry out a cyclopropanation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo cyclopropanation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·g_(cdw) ⁻¹, wherein g_(cdw) is the mass of cell dry weight in grams. For example, the turnover can be from about 0.1 to about 10 mmol·g_(cdw) ⁻¹, or from about 1 to about 10 mmol·g_(cdw) ⁻¹, or from about 5 to about 10 mmol or from about 0.01 to about 1 mmol·g_(cdw) ⁻¹, or from about 0.01 to about 0.1 mmol·g_(cdw) ⁻¹, or from about 0.1 to about 1 mmol·g_(cdw) ⁻¹, or greater than 1 mmol·g_(cdw) ⁻¹. The turnover can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10 mmol·g_(cdw) ⁻¹.

When whole cells expressing a heme enzyme are used to carry out a cyclopropanation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g g_(cdw) ⁻¹ h⁻¹). In general, in vivo cyclopropanation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g g_(cdw) ⁻¹ h⁻¹, wherein g_(cdw) is the mass of cell dry weight in grams. For example, the specific productivity can be from about 0.01 to about 0.1 g g_(cdw) ⁻¹ h⁻¹, or from about 0.1 to about 0.5 g g_(cdw) ⁻¹ h⁻¹, or greater than 0.5 g g_(cdw) ⁻¹ h⁻¹. The specific productivity can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or about 0.5 g g_(cdw) ⁻¹ h⁻¹.

In certain embodiments, mutations can be introduced into the target gene using standard cloning techniques (e.g., site-directed mutagenesis) or by gene synthesis to produce the heme enzymes (e.g., cytochrome P450 variants) of the present invention. The mutated gene can be expressed in a host cell (e.g., bacterial cell) using an expression vector under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Cyclopropanation activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein.

The expression vector comprising a nucleic acid sequence that encodes a heme enzyme of the invention can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage (e.g., a bacteriophage P1-derived vector (PAC)), a baculovirus vector, a yeast plasmid, or an artificial chromosome (e.g., bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC), and human artificial chromosome (HAC)). Expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequences. Equivalent expression vectors to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.

The expression vector can include a nucleic acid sequence encoding a heme enzyme that is operably linked to a promoter, wherein the promoter comprises a viral, bacterial, archaeal, fungal, insect, or mammalian promoter. In certain embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter.

It is understood that affinity tags may be added to the N- and/or C-terminus of a heme enzyme expressed using an expression vector to facilitate protein purification. Non-limiting examples of affinity tags include metal binding tags such as His6-tags and other tags such as glutathione S-transferase (GST).

Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017), pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene), pNH vectors, lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_(—)1 vectors, pChlamy_(—)1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1 (Promega, Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.). Non-limiting examples of expression vectors for use in eukaryotic host cells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors (Life Technologies), pKLAC1 vectors, pKLAC2 vectors (New England Biolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X™ adenoviral vectors (Clontech), and pBABE retroviral vectors. Any other vector may be used as long as it is replicable and viable in the host cell.

The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell.

Suitable bacterial host cells include, but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli M15, DH5α, DH10β, HB101, T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Chlamydomonas reinhardtii cells and Synechococcus elongates cells. Non-limiting examples of archaeal host cells include Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungal host cells include, but are not limited to, yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cells from the genera Aspergillus, Trichoderma, and Myceliophthora. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster. Non-limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.

In certain embodiments, the present invention provides heme enzymes such as the P450 variants described herein that are active cyclopropanation catalysts inside living cells. As a non-limiting example, bacterial cells (e.g., E. coli) can be used as whole cell catalysts for the in vivo cyclopropanation reactions of the present invention. In some embodiments, whole cell catalysts containing P450 enzymes with the equivalent C400X mutation are found to significantly enhance the total turnover number (TTN) compared to in vitro reactions using isolated P450 enzymes.

B. Compounds

The methods of the invention can be used to provide a number of cyclopropanation products. The cyclopropanation products include several classes of compound including, but not limited to, commodity and fine chemicals, flavors and scents, insecticides, and active ingredients in pharmaceutical compositions. The cyclopropanation products can also serve as starting materials or intermediates for the synthesis of compounds belonging to these and other classes.

In some embodiments, the cyclopropanation product is a compound according to formula I

For compounds of Formula I, R¹ is independently selected from H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(1a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃; and R² is independently selected from H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(2a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃. R^(1a) and R^(2a) are independently selected from H, optionally substituted C₁₋₁₈ alkyl and -L-R^(C).

When the moiety -L-R^(C) is present, L is selected from a bond, —C(R^(L))₂— and —NR^(L)—C(R^(L))₂—. Each R^(L) is independently selected from H, C₁₋₆ alkyl, halo, —CN, and —SO₂, and each R^(C) is selected from optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl.

For compounds of Formula I, R³, R⁴, R⁵, and R⁶ are independently selected from H, C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted C₁-C₆ alkoxy, halo, hydroxy, cyano, C(O)N(R⁷)₂, NR⁷C(O)R⁸, C(O)R⁸, C(O)OR⁸, and N(R⁹)₂. Each R⁷ and R⁸ is independently selected from H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl. Each R⁹ is independently selected from H, optionally substituted C₆₋₁₀ aryl, and optionally substituted 6- to 10-membered heteroaryl. Alternatively, two R⁹ moieties, together with the nitrogen atom to which they are attached, can form 6- to 18-membered heterocyclyl.

Alternatively, R³ forms an optionally substituted 3- to 18-membered ring with R⁴, or R⁵ forms an optionally substituted 18-membered ring with R⁶. R³ or R⁴ can also form a double bond with R⁵ or R⁶. R³ or R⁴ forms an optionally substituted 5- to 6-membered ring with R⁵ or R⁶.

In some embodiments, the cyclopropanation product is a compound of formula I as described above, wherein R¹ is C(O)O-L12^(c); R² is selected from H and optionally substituted C₆₋₁₀ aryl; and R³, R⁴, R⁵, and R⁶ are independently selected from H, optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, optionally substituted C₆₋₁₀ aryl, and halo. Alternatively, R³ can form an optionally substituted 3.to 18-membered ring with R⁴, or R⁵ can form an optionally substituted 3.to 18-membered ring with R⁶. In such embodiments, the cyclopropanation product can be a pyrethroid or a pyrethroid precursor.

In general, pyrethroids are characterized by an ester core having a structure according to Formula III:

Formula III is presented above as a cyclopropyl carboxylate moiety (“A”) esterified with an LR^(1c) moiety (“B”), with R^(1c) defined as for R^(C). The methods of the invention can be used to prepare pyrethroids and pyrethroid intermediates having a variety of “A” moieties connected to any of a variety of “B” moieties. For example, the pyrethroids can have an “A” moiety selected from:

For the A moieties listed above, X¹ is selected from H, optionally substituted C₁₋₆ alkyl, haloC₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, haloC₁₋₆ alkyl, optionally substituted C₁₋₆ alkylthio, C₁₋₆ alkylsilyl, halo, and cyano. X² is selected from H, chloro, and methyl. X³ is selected from H, methyl, halo, and CN. Each X⁴ is independently halo. Each X⁵ is independently selected from methyl and halo. X⁶ is selected from halo, optionally substituted C₁₋₆ alkyl, and optionally substituted C₁₋₆ alkoxy. X⁷ is selected from H, methyl, and halo. X⁸ is selected from H, halo, and optionally substituted C₁₋₆ alkyl. X⁹ is selected from H, halo, optionally substituted C₁₋₆ alkyl, C(O)O—(C₁₋₆ alkyl), C(O)—N(C₁₋₆ alkyl)₂, and cyano. Z¹, Z², and Z³ are independently selected from H, halo, optionally substituted C₁₋₆ alkyl, and optionally substituted C₆₋₁₀ aryl, or Z¹ and Z² are taken together to form an optionally substituted 5- to 6-membered cycloalkyl or heterocyclyl group. The wavy line at the right of each structure represents the point of connection between the A moiety and a B moiety.

The pyrethroids can have “B” moieties selected from:

For the B moieties listed above, each Y′ is independently selected from optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, phenyl, and (phenyl)C₁₋₆ alkoxy. Each Y² is independently selected from halo, optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₁₋₆ alkoxy, and nitro. Each Y³ is independently optionally substituted C₁₋₆ alkyl. Each Y⁴ is independently selected from optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, C₆₋₁₀ aryl-C₁₋₆ alkyl, furfuryl, C₁₋₆ alkoxy, (C₂₋₆ alkenyl)oxy, C₁₋₁₂ acyl, and halo. Y⁵ is selected from the group consisting of optionally substituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, and halo. The subscript m is an integer from 1 to 3, the subscript n is an integer from 1 to 5, the subscript p is an integer from 1 to 4, and the subscript q is an integer from 0 to 3. The wavy line at the left of each structure represents the point of connection between the B moiety and an A moiety.

The methods of the invention can be used to prepare pyrethroids having any A moiety joined to any B moiety. A given pyrethroid can have a structure selected from: A1-B1, A2-B1, A3-B1, A4-B1, A5-B1, A6-B1, A7-B1, A8-B1, A9-B1, A10-B1, A11-B1, A12-B1, A13-B1, A14-B1, A15-B1, A16-B1, A17-B1, A18-B1, A19-B1, A20-B1, A21-B1, A22-B1, A23-B1, A24-B1, A25-B1, A26-B1, A27-B1, A28-B1, A29-B1, A30-B1, A31-B1, A32-B1, A33-B1, A1-B2, A2-B2, A3-B2, A4-B2, A5-B2, A6-B2, A7-B2, A8-B2, A9-B2, A10-B2, A11-B2, A12-B2, A13-B2, A14-B2, A15-B2, A16-B2, A17-B2, A18-B2, A19-B2, A20-B2, A21-B2, A22-B2, A23-B2, A24-B2, A25-B2, A26-B2, A27-B2, A28-B2, A29-B2, A30-B2, A31-B2, A32-B2, A33-B2, A1-B3, A2-B3, A3-B3, A4-B3, A5-B3, A6-B3, A7-B3, A8-B3, A9-B3, A10-B3, A11-B3, A12-B3, A13-B3, A14-B3, A15-B3, A16-B3, A17-B3, A18-B3, A19-B3, A20-B3, A21-B3, A22-B3, A23-B3, A24-B3, A25-B3, A26-B3, A27-B3, A28-B3, A29-B3, A30-B3, A31-B3, A32-B3, A33-B3, A1-B4, A2-B4, A3-B4, A4-B4, A5-B4, A6-B4, A7-B4, A8-B4, A9-B4, A10-B4, A11-B4, A12-B4, A13-B4, A14-B4, A15-B4, A16-B4, A17-B4, A18-B4, A19-B4, A20-B4, A21-B4, A22-B4, A23-B4, A24-B4, A25-B4, A26-B4, A27-B4, A28-B4, A29-B4, A30-B4, A31-B4, A32-B4, A33-B4, A1-B5, A2-B5, A3-B5, A4-B5, A5-B5, A6-B5, A7-B5, A8-B5, A9-B5, A10-B5, A11-B5, A12-B5, A13-B5, A14-B5, A15-B5, A16-B5, A17-B5, A18-B5, A19-B5, A20-B5, A21-B5, A22-B5, A23-B5, A24-B5, A25-B5, A26-B5, A27-B5, A28-B5, A29-B5, A30-B5, A31-B5, A32-B5, A33-B5, A1-B6, A2-B6, A3-B6, A4-B6, A5-B6, A6-B6, A7-B6, A8-B6, A9-B6, A10-B6, A11-B6, A12-B6, A13-B6, A14-B6, A15-B6, A16-B6, A17-B6, A18-B6, A19-B6, A20-B6, A21-B6, A22-B6, A23-B6, A24-B6, A25-B6, A26-B6, A27-B6, A28-B6, A29-B6, A30-B6, A31-B6, A32-B6, A33-B6, A1-B7, A2-B7, A3-B7, A4-B7, A5-B7, A6-B7, A7-B7, A8-B7, A9-B7, A10-B7, A11-B7, A12-B7, A13-B7, A14-B7, A15-B7, A16-B7, A17-B7, A18-B7, A19-B7, A20-B7, A21-B7, A22-B7, A23-B7, A24-B7, A25-B7, A26-B7, A27-B7, A28-B7, A29-B7, A30-B7, A31-B7, A32-B7, A33-B7, A1-B8, A2-B8, A3-B8, A4-B8, A5-B8, A6-B8, A7-B8, A8-B8, A9-B8, A10-B8, A11-B8, A12-B8, A13-B8, A14-B8, A15-B8, A16-B8, A17-B8, A18-B8, A19-B8, A20-B8, A21-B8, A22-B8, A23-B8, A24-B8, A25-B8, A26-B8, A27-B8, A28-B8, A29-B8, A30-B8, A31-B8, A32-B8, A33-B8, A1-B9, A2-B9, A3-B9, A4-B9, A5-B9, A6-B9, A7-B9, A8-B9, A9-B9, A10-B9, A11-B9, A12-B9, A13-B9, A14-B9, A15-B9, A16-B9, A17-B9, A18-B9, A19-B9, A20-B9, A21-B9, A22-B9, A23-B9, A24-B9, A25-B9, A26-B9, A27-B9, A28-B9, A29-B9, A30-B9, A31-B9, A32-B9, A33-B9, A1-B10, A2-B10, A3-B10, A4-B10, A5-B10, A6-B10, A7-B10, A8-B10, A9-B10, A10-B10, A11-B10, A12-B10, A13-B10, A14-B10, A15-B10, A16-B10, A17-B10, A18-B10, A19-B10, A20-B10, A21-B10, A22-B10, A23-B10, A24-B10, A25-B10, A26-B10, A27-B10, A28-B10, A29-B10, A30-B10, A31-B10, A32-B10, A33-B10, A1-B11, A2-B11, A3-B11, A4-B11, A5-B11, A6-B11, A7-B11, A8-B11, A9-B11, A10-B11, A11-B11, A12-B11, A13-B11, A14-B11, A15-B11, A16-B11, A17-B11, A18-B11, A19-B11, A20-B11, A21-B11, A22-B11, A23-B11, A24-B11, A25-B11, A26-B11, A27-B11, A28-B11, A29-B11, A30-B11, A31-B11, A32-B11, A33-B11, A1-B12, A2-B12, A3-B12, A4-B12, A5-B12, A6-B12, A7-B12, A8-B12, A9-B12, A10-B12, A11-B12, A12-B12, A13-B12, A14-B12, A15-B12, A16-B12, A17-B12, A18-B12, A19-B12, A20-B12, A21-B12, A22-B12, A23-B12, A24-B12, A25-B12, A26-B12, A27-B12, A28-B12, A29-B12, A30-B12, A31-B12, A32-B12, A33-B12, A1-B13, A2-B13, A3-B13, A4-B13, A5-B13, A6-B13, A7-B13, A8-B13, A9-B13, A10-B13, A11-B13, A12-B13, A13-B13, A14-B13, A15-B13, A16-B13, A17-B13, A18-B13, A19-B13, A20-B13, A21-B13, A22-B13, A23-B13, A24-B13, A25-B13, A26-B13, A27-B13, A28-B13, A29-B13, A30-B13, A31-B13, A32-B13, A33-B13, A1-B14, A2-B14, A3-B14, A4-B14, A5-B14, A6-B14, A7-B14, A8-B14, A9-B14, A10-B14, A11-B14, A12-B14, A13-B14, A14-B14, A15-B14, A16-B14, A17-B14, A18-B14, A19-B14, A20-B14, A21-B14, A22-B14, A23-B14, A24-B14, A25-B14, A26-B14, A27-B14, A28-B14, A29-B14, A30-B14, A31-B14, A32-B14, A33-B14, A1-B15, A2-B15, A3-B15, A4-B15, A5-B15, A6-B15, A7-B15, A8-B15, A9-B15, A10-B15, A11-B15, A12-B15, A13-B15, A14-B15, A15-B15, A16-B15, A17-B15, A18-B15, A19-B15, A20-B15, A21-B15, A22-B15, A23-B15, A24-B15, A25-B15, A26-B15, A27-B15, A28-B15, A29-B15, A30-B15, A31-B15, A32-B15, A33-B15, A1-B16, A2-B16, A3-B16, A4-B16, A5-B16, A6-B16, A7-B16, A8-B16, A9-B16, A10-B16, A11-B16, A12-B16, A13-B16, A14-B16, A15-B16, A16-B16, A17-B16, A18-B16, A19-B16, A20-B16, A21-B16, A22-B16, A23-B16, A24-B16, A25-B16, A26-B16, A27-B16, A28-B16, A29-B16, A30-B16, A31-B16, A32-B16, A33-B16, A1-B17, A2-B17, A3-B17, A4-B17, A5-B17, A6-B17, A7-B17, A8-B17, A9-B17, A10-B17, A11-B17, A12-B17, A13-B17, A14-B17, A15-B17, A16-B17, A17-B17, A18-B17, A19-B17, A20-B17, A21-B17, A22-B17, A23-B17, A24-B17, A25-B17, A26-B17, A27-B17, A28-B17, A29-B17, A30-B17, A31-B17, A32-B17, A33-B17, A1-B18, A2-B18, A3-B18, A4-B18, A5-B18, A6-B18, A7-B18, A8-B18, A9-B18, A10-B18, A11-B18, A12-B18, A13-B18, A14-B18, A15-B18, A16-B18, A17-B18, A18-B18, A19-B18, A20-B18, A21-B18, A22-B18, A23-B18, A24-B18, A25-B18, A26-B18, A27-B18, A28-B18, A29-B18, A30-B18, A31-B18, A32-B18, A33-B18, A1-B19, A2-B19, A3-B19, A4-B19, A5-B19, A6-B19, A7-B19, A8-B19, A9-B19, A10-B19, A11-B19, A12-B19, A13-B19, A14-B19, A15-B19, A16-B19, A17-B19, A18-B19, A19-B19, A20-B19, A21-B19, A22-B19, A23-B19, A24-B19, A25-B19, A26-B19, A27-B19, A28-B19, A29-B19, A30-B19, A31-B19, A32-B19, A33-B19, A1-B20, A2-B20, A3-B20, A4-B20, A5-B20, A6-B20, A7-B20, A8-B20, A9-B20, A10-B20, A11-B20, A12-B20, A13-B20, A14-B20, A15-B20, A16-B20, A17-B20, A18-B20, A19-B20, A20-B20, A21-B20, A22-B20, A23-B20, A24-B20, A25-B20, A26-B20, A27-B20, A28-B20, A29-B20, A30-B20, A31-B20, A32-B20, A33-B20, A1-B21, A2-B21, A3-B21, A4-B21, A5-B21, A6-B21, A7-B21, A8-B21, A9-B21, A10-B21, A11-B21, A12-B21, A13-B21, A14-B21, A15-B21, A16-B21, A17-B21, A18-B21, A19-B21, A20-B21, A21-B21, A22-B21, A23-B21, A24-B21, A25-B21, A26-B21, A27-B21, A28-B21, A29-B21, A30-B21, A31-B21, A32-B21, A33-B21, A1-B22, A2-B22, A3-B22, A4-B22, A5-B22, A6-B22, A7-B22, A8-B22, A9-B22, A10-B22, A11-B22, A12-B22, A13-B22, A14-B22, A15-B22, A16-B22, A17-B22, A18-B22, A19-B22, A20-B22, A21-B22, A22-B22, A23-B22, A24-B22, A25-B22, A26-B22, A27-B22, A28-B22, A29-B22, A30-B22, A31-B22, A32-B22, A33-B22, A1-B23, A2-B23, A3-B23, A4-B23, A5-B23, A6-B23, A7-B23, A8-B23, A9-B23, A10-B23, A11-B23, A12-B23, A13-B23, A14-B23, A15-B23, A16-B23, A17-B23, A18-B23, A19-B23, A20-B23, A21-B23, A22-B23, A23-B23, A24-B23, A25-B23, A26-B23, A27-B23, A28-B23, A29-B23, A30-B23, A31-B23, A32-B23, A33-B23, A1-B24, A2-B24, A3-B24, A4-B24, A5-B24, A6-B24, A7-B24, A8-B24, A9-B24, A10-B24, A11-B24, A12-B24, A13-B24, A14-B24, A15-B24, A16-B24, A17-B24, A18-B24, A19-B24, A20-B24, A21-B24, A22-B24, A23-B24, A24-B24, A25-B24, A26-B24, A27-B24, A28-B24, A29-B24, A30-B24, A31-B24, A32-B24, A33-B24, A1-B25, A2-B25, A3-B25, A4-B25, A5-B25, A6-B25, A7-B25, A8-B25, A9-B25, A10-B25, A11-B25, A12-B25, A13-B25, A14-B25, A15-B25, A16-B25, A17-B25, A18-B25, A19-B25, A20-B25, A21-B25, A22-B25, A23-B25, A24-B25, A25-B25, A26-B25, A27-B25, A28-B25, A29-B25, A30-B25, A31-B25, A32-B25, A33-B25, A1-B26, A2-B26, A3-B26, A4-B26, A5-B26, A6-B26, A7-B26, A8-B26, A9-B26, A10-B26, A11-B26, A12-B26, A13-B26, A14-B26, A15-B26, A16-B26, A17-B26, A18-B26, A19-B26, A20-B26, A21-B26, A22-B26, A23-B26, A24-B26, A25-B26, A26-B26, A27-B26, A28-B26, A29-B26, A30-B26, A31-B26, A32-B26, A33-B26, A1-B27, A2-B27, A3-B27, A4-B27, A5-B27, A6-B27, A7-B27, A8-B27, A9-B27, A10-B27, A11-B27, A12-B27, A13-B27, A14-B27, A15-B27, A16-B27, A17-B27, A18-B27, A19-B27, A20-B27, A21-B27, A22-B27, A23-B27, A24-B27, A25-B27, A26-B27, A27-B27, A28-B27, A29-B27, A30-B27, A31-B27, A32-B27, A33-B27, A1-B28, A2-B28, A3-B28, A4-B28, A5-B28, A6-B28, A7-B28, A8-B28, A9-B28, A10-B28, A11-B28, A12-B28, A13-B28, A14-B28, A15-B28, A16-B28, A17-B28, A18-B28, A19-B28, A20-B28, A21-B28, A22-B28, A23-B28, A24-B28, A25-B28, A26-B28, A27-B28, A28-B28, A29-B28, A30-B28, A31-B28, A32-B28, A33-B28, A1-B29, A2-B29, A3-B29, A4-B29, A5-B29, A6-B29, A7-B29, A8-B29, A9-B29, A10-B29, A11-B29, A12-B29, A13-B29, A14-B29, A15-B29, A16-B29, A17-B29, A18-B29, A19-B29, A20-B29, A21-B29, A22-B29, A23-B29, A24-B29, A25-B29, A26-B29, A27-B29, A28-B29, A29-B29, A30-B29, A31-B29, A32-B29, A33-B29, A1-B30, A2-B30, A3-B30, A4-B30, A5-B30, A6-B30, A7-B30, A8-B30, A9-B30, A10-B30, A11-B30, A12-B30, A13-B30, A14-B30, A15-B30, A16-B30, A17-B30, A18-B30, A19-B30, A20-B30, A21-B30, A22-B30, A23-B30, A24-B30, A25-B30, A26-B30, A27-B30, A28-B30, A29-B30, A30-B30, A31-B30, A32-B30, A33-B30, A1-B31, A2-B31, A3-B31, A4-B31, A5-B31, A6-B31, A7-B31, A8-B31, A9-B31, A10-B31, A11-B31, A12-B31, A13-B31, A14-B31, A15-B31, A16-B31, A17-B31, A18-B31, A19-B31, A20-B31, A21-B31, A22-B31, A23-B31, A24-B31, A25-B31, A26-B31, A27-B31, A28-B31, A29-B31, A30-B31, A31-B31, A32-B31, A33-B31, A1-B32, A2-B32, A3-B32, A4-B32, A5-B32, A6-B32, A7-B32, A8-B32, A9-B32, A10-B32, A11-B32, A12-B32, A13-B32, A14-B32, A15-B32, A16-B32, A17-B32, A18-B32, A19-B32, A20-B32, A21-B32, A22-B32, A23-B32, A24-B32, A25-B32, A26-B32, A27-B32, A28-B32, A29-B32, A30-B32, A31-B32, A32-B32, A33-B32, A1-B33, A2-B33, A3-B33, A4-B33, A5-B33, A6-B33, A7-B33, A8-B33, A9-B33, A10-B33, A11-B33, A12-B33, A13-B33, A14-B33, A15-B33, A16-B33, A17-B33, A18-B33, A19-B33, A20-B33, A21-B33, A22-B33, A23-B33, A24-B33, A25-B33, A26-B33, A27-B33, A28-B33, A29-B33, A30-B33, A31-B33, A32-B33, A33-B33, A1-B34, A2-B34, A3-B34, A4-B34, A5-B34, A6-B34, A7-B34, A8-B34, A9-B34, A10-B34, A11-B34, A12-B34, A13-B34, A14-B34, A15-B34, A16-B34, A17-B34, A18-B34, A19-B34, A20-B34, A21-B34, A22-B34, A23-B34, A24-B34, A25-B34, A26-B34, A27-B34, A28-B34, A29-B34, A30-B34, A31-B34, A32-B34, A33-B34, A1-B35, A2-B35, A3-B35, A4-B35, A5-B35, A6-B35, A7-B35, A8-B35, A9-B35, A10-B35, A11-B35, A12-B35, A13-B35, A14-B35, A15-B35, A16-B35, A17-B35, A18-B35, A19-B35, A20-B35, A21-B35, A22-B35, A23-B35, A24-B35, A25-B35, A26-B35, A27-B35, A28-B35, A29-B35, A30-B35, A31-B35, A32-B35, A33-B35, A1-B36, A2-B36, A3-B36, A4-B36, A5-B36, A6-B36, A7-B36, A8-B36, A9-B36, A10-B36, A11-B36, A12-B36, A13-B36, A14-B36, A15-B36, A16-B36, A17-B36, A18-B36, A19-B36, A20-B36, A21-B36, A22-B36, A23-B36, A24-B36, A25-B36, A26-B36, A27-B36, A28-B36, A29-B36, A30-B36, A31-B36, A32-B36, A33-B36, A1-B37, A2-B37, A3-B37, A4-B37, A5-B37, A6-B37, A7-B37, A8-B37, A9-B37, A10-B37, A11-B37, A12-B37, A13-B37, A14-B37, A15-B37, A16-B37, A17-B37, A18-B37, A19-B37, A20-B37, A21-B37, A22-B37, A23-B37, A24-B37, A25-B37, A26-B37, A27-B37, A28-B37, A29-B37, A30-B37, A31-B37, A32-B37, A33-B37, A1-B38, A2-B38, A3-B38, A4-B38, A5-B38, A6-B38, A7-B38, A8-B38, A9-B38, A10-B38, A11-B38, A12-B38, A13-B38, A14-B38, A15-B38, A16-B38, A17-B38, A18-B38, A19-B38, A20-B38, A21-B38, A22-B38, A23-B38, A24-B38, A25-B38, A26-B38, A27-B38, A28-B38, A29-B38, A30-B38, A31-B38, A32-B38, A33-B38, A1-B39, A2-B39, A3-B39, A4-B39, A5-B39, A6-B39, A7-B39, A8-B39, A9-B39, A10-B39, A11-B39, A12-B39, A13-B39, A14-B39, A15-B39, A16-B39, A17-B39, A18-B39, A19-B39, A20-B39, A21-B39, A22-B39, A23-B39, A24-B39, A25-B39, A26-B39, A27-B39, A28-B39, A29-B39, A30-B39, A31-B39, A32-B39, and A33-B39. The A moiety is joined to the B moiety to form the ester bond as shown in Formula III above.

A pyrethroid prepared according to the methods of the invention can have, for example, a structure selected from:

Z¹, Z², and Z³ are independently selected from H, halo, optionally substituted C₁₋₆ alkyl, and optionally substituted C₆₋₁₀ aryl. Z¹ and Z² can also be taken together to form an optionally substituted 5- to 6-membered cycloalkyl or heterocyclyl group.

In some embodiments, the methods of the invention can be used to prepare pyrethroid intermediate compounds that can be converted to the pyrethroid compounds described above. Alkyl esters of cyclopropanecarboxylic acid and cyclopropanecarboxylic acid derivatives can be converted to a variety of pyrethroid compounds via reaction with appropriate alcohols.

Accordingly, some embodiments of the invention provide methods as wherein the cyclopropanation product is a compound according to formula II:

For compounds of formula II: R^(1a) is C₁₋₆ alkyl and R² is selected from H and optionally substituted C₆₋₁₀ aryl. In some embodiments, R² is H. In some embodiments, the compound of formula II is selected from:

In such embodiments, X′ is selected from H, optionally substituted C₁₋₆ alkyl, haloC₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, optionally substituted C₁₋₆ alkylthio, C₁₋₆ alkylsilyl, halo, and cyano. X² is selected from H, chloro, and methyl. X³ is selected from H, methyl, halo, and CN. Each X⁴ is independently halo. Each X⁵ is independently selected from methyl and halo. X⁶ is selected from halo, optionally substituted C₁₋₆ alkyl, and optionally substituted C₁₋₆ alkoxy. X⁷ is selected from H, methyl, and halo. X⁸ is selected from H, halo, and optionally substituted C₁₋₆ alkyl. X⁹ is selected from H, halo, optionally substituted C₁₋₆ alkyl, C(O)O—(C₁₋₆ alkyl), C(O)—N(C₁₋₆ alkyl)₂, and cyano. Z′, Z², and Z³ are independently selected from H, halo, optionally substituted C₁₋₆ alkyl, and optionally substituted C₆₋₁₀ aryl. Z′ and Z² can also be taken together to form an optionally substituted 5- to 6-membered cycloalkyl or heterocyclyl group

In some embodiments, the methods of the invention include converting the cyclopropanation product according to formula II to a compound according to formula III:

For compounds of formula III, L is selected from a bond, —C(R^(L))₂—, and —NR^(L)—C(R^(L))₂—. Each R^(L) is independently selected from H, —CN, and —SO₂. R^(1C) is selected from optionally subsistuted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl. In some embodiments, L in the compounds of formula III is selected from a bond, —CH₂—, —CH(CN)—, and —N(SO₂)—CH₂.

In some embodiments, the moiety L-R^(1C) in the compounds according to formula III has a structure selected from:

In such embodiments, each Y¹ is independently selected from optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, phenyl, and (phenyl)C₁₋₆ alkoxy. Each Y² is independently selected from halo, optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₁₋₆ alkoxy, and nitro. Each Y³ is independently optionally substituted C₁₋₆ alkyl. Each Y⁴ is independently selected from optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, C₆₋₁₀ aryl-C₁₋₆ alkyl, furfuryl, C₁₋₆ alkoxy, (C₂₋₆ alkenyl)oxy, C₁₋₁₂ acyl, and halo. Y⁵ is selected from the group consisting of optionally substituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, and halo. The subscript m is an integer from 1 to 3, the subscript n is an integer from 1 to 5, the subscript p is an integer from 1 to 4, and the subscript q is an integer from 0 to 3. The wavy line at the left of each structure represents the point of connection between the moiety -L-R^(1c) and the rest of the compound according to formula III.

In some embodiments, the compound of formula III is selected from:

In some embodiments, the compound of formula III is resmethrin.

As for the pyrethroids discussed herein, a number of other compounds can be synthesized via processes that include a cyclopropanation product. Such processes are generalized in Scheme 1 showing the enzyme-catalyzed formation of a cyclopropanation product from an olefinic substrate and a diazo reagent, followed by chemical conversion of to a final product such as a pharmaceutical agent. Depending on the particular final product, the process can include conversion of the cyclopropanation product to one or more synthetic intermediates prior to preparation of the final product. Non-limiting examples of cyclopropanation products useful in such processes are summarized in Table 6.

TABLE 6 Cyclopropanation for synthesis of intermediates en route to biologically active compounds. Cyclopropanation Final Olefinic Substrate Diazo Reagent Product/Intermediate Product

milnacipran

milnacipran

milnacipran; bicifidine; 1-(3,4- dichloro- phenyl)-3- azabicyclo [3.1.0] hexane

milnacipran; bicifidine; 1-(3,4- dichloro- phenyl)-3- azabicyclo [3.1.0] hexane

cilastain

boceprevir

boceprevir

boceprevir

1R,2S- fluoro- cyclopropyl- amine, sitafloxacin

1R,2S- fluoro- cyclopropyl- amine, sitafloxacin

antho- plalone, norantho- plone

odanacatib

odanacatib

montekulast

montekulast

carene

pyrethrin II

In some embodiments, the cyclopropanation product is a compound having a structure according to the formula:

wherein R^(1a) is optionally substituted C₁₋₆ alkyl, and R⁵ and R⁶ are independently selected from H, optionally substituted C₁₋₆ alkyl, optionally substituted C₆₋₁₀ aryl, C(O)N(R⁷)₂, C(O)OR⁸ and NR⁷C(O)R⁸.

In some embodiments, the cyclopropanation product has the structure selected from:

In such embodiments, the methods of the invention can include converting the cyclopropanation product to a compound selected from milcanipran, levomilnacipran, bicifadine, and 1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0]hexane.

The methods of the invention can be used to prepare several different types of compounds having cyclopropane functional groups. The compounds include, but are not limited to, pharmaceutical agents having chiral cyclopropane moieties, pharmaceutical agents having achiral cyclopropane moieties, insecticides, plant hormones, flavors, scents, and fatty acids.

In some embodiments, the methods of the invention are used to prepare a compound selected from:

Synthesis of prostratin, a protein kinase C activator, is shown as a non-limiting example in Scheme 2. The prostratin cyclopropane moiety can be installed by heme enzyme-catalyzed intramolecular or intermolecular cyclopropanation reactions.

Some embodiments of the invention provide a method as described above, wherein the olefinic substrate is selected from the group consisting of an alkene, a cycloalkane, and an arylalkene. In some embodiments, the olefinic substrate is an arylalkene. In some embodiments, the arylalkene is a styrene.

In some embodiments, the styrene has the formula:

R³ is selected from the H, optionally substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, C(O)N(R⁷)₂, C(O)OR⁸, N(R⁹)₂, halo, hydroxy, and cyano. R⁵ and R⁶ are independently selected from H, optionally substituted C₁₋₆ alkyl, and halo. R¹⁰ is selected from optionally substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, halo, and haloalkyl, and the subscript r is an integer from 0 to 2.

In general, the diazo reagents useful in the methods of the invention have the structure:

wherein R¹ and R² are defined as for the cyclopropanation products. Any diazoreagent can be added to the reaction as a reagent itself, or the diazoreagent can be prepared in situ.

In some embodiments, the diazo reagent is selected from an α-diazoester, an α-diazoamide, an α-diazonitrile, an α-diazoketone, an α-diazoaldehyde, and an α-diazosilane. In some embodiments, the diazo reagent has a formula selected from:

wherein R^(1a) is selected from H and optionally substituted C₁-C₆ alkyl; and each R⁷ and R⁸ is independently selected from H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl.

In some embodiments, the diazo reagent is selected from the group consisting of diazomethane, ethyl diazoacetate, and (trimethylsilyl)diazomethane.

In some embodiments, the diazo reagent is an α-diazoester. In some embodiments, the diazo reagent has the formula:

In some embodiments, the cyclopropanation product has a formula selected from:

One of skill in the art will appreciate that stereochemical configuration of the cyclopropanation product will be determined in part by the orientation of the diazo reagent with respect to the position of an olefinic substrate such as styrene during the cyclopropanation step. For example, any substituent originating from the olefinic substrate can be positioned on the same side of the cyclopropyl ring as a substituent origination from the diazo reagent. Cyclopropanation products having this arrangement are called “cis” compounds or “Z” compounds. Any substituent originating from the olefinic substrate and any substituent originating from the diazo reagent can also be on opposite sides of the cyclopropyl ring. Cyclopropanation products having this arrangement are called “trans” compounds or “E” compounds. An example of such arrangments is shown in the reaction scheme of FIG. 29.

As shown in FIG. 29, two cis isomers and two trans isomers can arise from the reaction of an olefinic substrate with a diazo reagent. The two cis isomers are enantiomers with respect to one another, in that the structures are non-superimposable mirror images of each other. Similarly, the two trans isomers are enantiomers. One of skill in the art will appreciate that the absolute stereochemistry of a cyclopropranation product—that is, whether a given chiral center exhibits the right-handed “R” configuration or the left-handed “S” configuration—will depend on factors including the structures of the particular olefinic substrate and diazo reagent used in the reaction, as well as the identity of the enzyme. This is also true for the relative stereochemistry—that is, whether a cyclopropanation product exhibits a cis or trans configuration—as well as for the distribution of cyclopropanation product mixtures will also depend on such factors.

In general, cyclopropanation product mixtures have cis:trans ratios ranging from about 1:99 to about 99:1. The cis:trans ratio can be, for example, from about 1:99 to about 1:75, or from about 1:75 to about 1:50, or from about 1:50 to about 1:25, or from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The cis:trans ratio can be from about 1:80 to about 1:20, or from about 1:60 to about 1:40, or from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The cis:trans ratio can be about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, or about 1:95. The cis:trans ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.

The distribution of a cyclopropanation product mixture can be assessed in terms of the enantiomeric excess, or “% ee,” of the mixture. The enantiomeric excess refers to the difference in the mole fractions of two enantiomers in a mixture. Taking the reaction scheme in FIG. 29 as a non-limiting example, for instance, the enantiomeric excess of the “E” or trans (R,R) and (S,S) enantiomers can be calculated using the formula: % ee_(E)=[(χ_(R,R)−χ_(S,X))/(χ_(R,R)+χ_(S,S))]×100%, wherein χ is the mole fraction for a given enantiomer. The enantiomeric excess of the “Z” or cis enantiomers (% ee_(z)) can be calculated in the same manner.

In general, cyclopropanantion product mixtures exhibit % ee values ranging from about 1% to about 99%, or from about −1% to about −99%. The closer a given % ee value is to 99% (or −99%), the purer the reaction mixture is. The % ee can be, for example, from about −90% to about 90%, or from about −80% to about 80%, or from about −70% to about 70%, or from about −60% to about 60%, or from about −40% to about 40%, or from about −20% to about 20%. The % ee can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The % ee can be from about −1% to about −99%, or from about −20% to about −80%, or from about −40% to about −60%, or from about −1% to about −25%, or from about −25% to about −50%, or from about −50% to about −75%. The % ee can be about −99%, −95%, −90%, −85%, −80%, −75%, −70%, −65%, −60%, −55%, −50%, −45%, −40%, −35%, −30%, −25%, −20%, −15%, −10%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95%. Any of these values can be % ee_(E) values or % ee_(z) values.

Accordingly, some embodiments of the invention provide methods for producing a plurality of cyclopropanation products having a % ee_(z) of from about −90% to about 90%. In some embodiments, the % ee_(z) is at least 90%. In some embodiments, the % ee_(z) is at least −99%. In some embodiments, the % ee_(E) is from about −90% to about 90%. In some embodiments, the % ee_(E) is at least 90%. In some embodiments, the % ee_(E) is at least −99%.

In a related aspect, certain embodiments of the invention provide cyclopropane-containing compounds according to any of Formulas I, II, III as described herein. The compounds are prepared using the methods of the invention. In some embodiments, the invention provides a pyrethroid prepared according to the methods of the invention. In some embodiments, the invention provides milnacipran, levomilnacipran, bicifadine, or 1-(3,4-dichlorophenyl)-3-azabicyclo[3.1.0]hexane prepared according to the methods of the invention. In some embodiments, the invention provides any of the compounds illustrated in Table 6, which compounds are prepared according to the methods of the invention. The invention can provide other compounds prepared according to the methods described herein.

C. Reaction Conditions

The methods of the invention include forming reaction mixtures that contain the heme enzymes described herein. The heme enzymes can be, for example, purified prior to addition to a reaction mixture or secreted by a cell present in the reaction mixture. The reaction mixture can contain a cell lysate including the enzyme, as well as other proteins and other cellular materials. Alternatively, a heme enzyme can catalyze the reaction within a cell expressing the heme enzyme. Any suitable amount of heme enzyme can be used in the methods of the invention. In general, cyclopropanation reaction mixtures contain from about 0.01 mol % to about 10 mol % heme enzyme with respect to the diazo reagent and/or olefinic substrate. The reaction mixtures can contain, for example, from about 0.01 mol % to about 0.1 mol % heme enzyme, or from about 0.1 mol % to about 1 mol % heme enzyme, or from about 1 mol % to about 10 mol % heme enzyme. The reaction mixtures can contain from about 0.05 mol % to about 5 mol % heme enzyme, or from about 0.05 mol % to about 0.5 mol % heme enzyme. The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1 mol % heme enzyme.

The concentration of olefinic substrate and diazo reagent are typically in the range of from about 100 μM to about 1 M. The concentration can be, for example, from about 100 μM to about 1 mM, or about from 1 mM to about 100 mM, or from about 100 mM to about 500 mM, or from about 500 mM to 1 M. The concentration can be from about 500 μM to about 500 mM, 500 μM to about 50 mM, or from about 1 mM to about 50 mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30 mM. The concentration of olefinic substrate or diazo reagent can be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μM. The concentration of olefinic substrate or diazo reagent can be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

Reaction mixtures can contain additional reagents. As non-limiting examples, the reaction mixtures can contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 3-morpholinopropane-1-sulfonic acid (MOPS), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate, sodium phosphate, phosphate-buffered saline, sodium citrate, sodium acetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide, dimethylformamide, ethanol, methanol, isopropanol, glycerol, tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g., NaCl, KCl, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., urea and guandinium hydrochloride), detergents (e.g., sodium dodecylsulfate and Triton-X 100), chelators (e.g., ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2-({2-[Bis(carboxymethyl]amino)ethyl}(carboxymethyl)amino)acetic acid (EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducing agents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)). Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents can be used at any suitable concentration, which can be readily determined by one of skill in the art. In general, buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents, if present, are included in reaction mixtures at concentrations ranging from about 1 μM to about 1 M. For example, a buffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, a sugar, or a reducing agent can be included in a reaction mixture at a concentration of about 1 or about 10 μM, or about 100 μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M. In some embodiments, a reducing agent is used in a sub-stoichiometric amount with respect to the olefin substrate and the diazo reagent. Cosolvents, in particular, can be included in the reaction mixtures in amounts ranging from about 1% v/v to about 75% v/v, or higher. A cosolvent can be included in the reaction mixture, for example, in an amount of about 5, 10, 20, 30, 40, or 50% (v/v).

Reactions are conducted under conditions sufficient to catalyze the formation of a cyclopropanation product. The reactions can be conducted at any suitable temperature. In general, the reactions are conducted at a temperature of from about 4° C. to about 40° C. The reactions can be conducted, for example, at about 25° C. or about 37° C. The reactions can be conducted at any suitable pH. In general, the reactions are conducted at a pH of from about 6 to about 10. The reactions can be conducted, for example, at a pH of from about 6.5 to about 9. The reactions can be conducted for any suitable length of time. In general, the reaction mixtures are incubated under suitable conditions for anywhere between about 1 minute and several hours. The reactions can be conducted, for example, for about 1 minute, or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 72 hours. Reactions can be conducted under aerobic conditions or anaerobic conditions. Reactions can be conducted under an inert atmosphere, such as a nitrogen atmosphere or argon atmosphere. In some embodiments, a solvent is added to the reaction mixture. In some embodiments, the solvent forms a second phase, and the cyclopropanation occurs in the aqueous phase. In some embodiments, the heme enzymes is located in the aqueous layer whereas the substrates and/or products occur in an organic layer. Other reaction conditions may be employed in the methods of the invention, depending on the identity of a particular heme enzyme, olefinic substrate, or diazo reagent.

Reactions can be conducted in vivo with intact cells expressing a heme enzyme of the invention. The in vivo reactions can be conducted with any of the host cells used for expression of the heme enzymes, as described herein. A suspension of cells can be formed in a suitable medium supplemented with nutrients (such as mineral micronutrients, glucose and other fuel sources, and the like). Cyclopropanation yields from reactions in vivo can be controlled, in part, by controlling the cell density in the reaction mixtures. Cellular suspensions exhibiting optical densities ranging from about 0.1 to about 50 at 600 nm can be used for cyclopropanation reactions. Other densisties can be useful, depending on the cell type, specific heme enzymes, or other factors.

The methods of the invention can be assessed in terms of the diastereoselectivity and/or enantioselectivity of cyclopropanation reaction—that is, the extent to which the reaction produces a particular isomer, whether a diastereomer or enantiomer. A perfectly selective reaction produces a single isomer, such that the isomer constitutes 100% of the product. As another non-limiting example, a reaction producing a particular enantiomer constituting 90% of the total product can be said to be 90% enantioselective. A reaction producing a particular diastereomer constituting 30% of the total product, meanwhile, can be said to be 30% diastereoselective.

In general, the methods of the invention include reactions that are from about 1% to about 99% diastereoselective. The reactions are from about 1% to about 99% enantioselective. The reaction can be, for example, from about 10% to about 90% diastereoselective, or from about 20% to about 80% diastereoselective, or from about 40% to about 60% diastereoselective, or from about 1% to about 25% diastereoselective, or from about 25% to about 50% diastereoselective, or from about 50% to about 75% diastereoselective. The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% diastereoselective. The reaction can be from about 10% to about 90% enantioselective, from about 20% to about 80% enantioselective, or from about 40% to about 60% enantioselective, or from about 1% to about 25% enantioselective, or from about 25% to about 50% enantioselective, or from about 50% to about 75% enantioselective. The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% enantioselective. Accordingly some embodiments of the invention provide methods wherein the reaction is at least 30% to at least 90% diastereoselective. In some embodiments, the reaction is at least 30% to at least 90% enantioselective.

IV. EXAMPLES

The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1 C═C Functionalization by Enzyme-Catalyzed Carbenoid Insertion

This example illustrates bacterial cytochrome P450s that are engineered to catalyze highly stereoselective carbene transfers to aryl-substituted olefins, a reaction without a known biological counterpart.

Creating enzymes that catalyze novel reactions, one of the hallmarks of evolution, is a huge challenge and nearly unexplored frontier in protein engineering. Carbene transfers to C═C bonds are powerful catalytic methods that lack biological counterparts. Stereo-control over these transformations currently relies on expensive transition metal catalysts that require toxic organic solvents and are difficult to systematically modify or optimize. This example illustrates variants of cytochrome P450_(BM3) that catalyze an important reaction not previously known for this monooxygenase: the cyclopropanation of styrene from diazoester reagents with exquisite enantio- and diastereocontrol. As such, this example demonstrates that existing enzymes can be adapted for catalysis of synthetically-important reactions not previously observed in Nature.

Introduction

The many strategies for functionalizing C═C bonds that have evolved in the biological world have captivated the imaginations of chemists who attempt to develop ‘biomimetic’ catalysts (J. T. Groves, Proc. Natl. Acad. Sci. U.S.A. 100, 3569 (2003); R. Breslow, J. Biol. Chem. 284, 1337 (2009)). The reverse of this, developing new biocatalysts inspired by synthetic chemistry, has received little attention, mainly because we poorly understand how to encode a desired function in a protein sequence. Nature's entire catalyst repertoire has been built with and utilizes physiologically accessible reagents. Not subject to the same limitations, synthetic chemists have developed powerful methods for direct C═C functionalization based on transition metal-catalyzed carbenoid insertions, reactions that are used extensively in the synthesis of natural product intermediates and artificial drugs (H. M. L. Davies et al., Nature 451, 417 (2008)). Utilizing high-energy precursors typically in the form of acceptor-substituted diazo reagents, these synthetic systems, upon dinitrogen extrusion, form metallo-carbenoid intermediates that insert into C═C bonds to form new carbon-carbon centers. Synthetic catalysts, however, require expensive transition metals and elaborate ligand designs for stereocontrol; they also often require toxic organic solvents. This example demonstrates combining the high levels of selectivity and ‘green’ process conditions afforded by enzymes with the synthetic power of carbene transfer strategies enabled by transition metal catalysis.

Results

Members of the diverse cytochrome P450 enzyme family catalyze myriad oxidative transformations, including hydroxylation, epoxidation, oxidative ring coupling, heteratom release, and heteroatom oxygenation (E. M. Isin et al., Biochim. Biophys. Acta 1770, 314 (2007)). The majority of transformations encompassed by this broad catalytic scope are manifestations of the same high-valent iron-oxene intermediate (compound I, FIG. 1). Inspired by the impressive chemo-, regio- and stereo-selectivities with which cytochrome P450s can insert oxygen atoms into C═C bonds, whether these enzymes could be engineered to mimic this chemistry was investigated for isoelectronic carbene transfer reactions via high-valent iron-enoid species (FIG. 1). This example shows that variants of the cytochrome P450 from Bacillus megaterium (CYP102A1, or P450_(BM3)) are efficient catalysts for the asymmetric metallocarbene-mediated cyclopropanation of styrenes.

Since iron porphyrins are known to catalyze carbene-based cyclopropanations (J. R. Wolf et al., J. Am. Chem. Soc. 117, 9194 (1995)), whether some common heme proteins display measurable levels of ‘cyclopropanase’ activity was first probed. The reaction between styrene and ethyl diazoacetate (EDA, FIG. 2), a well-recognized model system for validating new cyclopropanation catalysts, was investigated. Initial experiments showed that optimal formation of the desired cyclopropanation products occurred in water in the presence of a reducing agent (e.g., sodium dithionite) under anaerobic conditions (Tables 7-10). Horseradish peroxidase (HRP), cytochrome c (cyt c), myoglobin (Mb) and P450_(BM3) all displayed multiple turnovers towards the cyclopropane products, with HRP, cyt c and Mb showing negligible enantio-induction and forming the trans cyclopropane with over 90% selectivity, which is comparable to the diastereoselectivity induced by free hemin (Table 7). Interestingly, P450_(BM3), despite forming the cyclopropane products in low yield, catalyzed the reaction with different diasteroselectivity (cis:trans 37:63) and slight enantio-induction (Table 11), indicating that carbene transfer and selectivity were dictated by the active site-bound heme cofactor rather than by hemin released from the protein.

TABLE 7 Heme catalysts under anaerobic conditions with sodium dithionite (Na₂S₂O₄).

Cat. loading Catalyst Axial ligand (% mol eq) TTN cis:trans^(a) % ee cis^(b) % ee trans^(c) Catalase O-Tyr 0.16 0 — — — CPO^(d) S-Cys 0.40 0 — — — HRP N-His 1.00 9 7:93 8 −7 cyt c N-His, S-Met 1.00 19 6:94 0 12 Mb N-His 1.00 43 6:94 −1 2 P450_(BM3) S-Cys 0.20 5 37:63  −27 −2 Hemin — 0.20 73 6:94 −1 0 ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,R) - (S,S). ^(d)Bioconversion conducted at 0.1 M citrate buffer pH = 4.0.

TABLE 8 Heme catalysts under anaerobic conditions without Na₂S₂O₄.

Cat. loading Catalyst Axial ligand (% mol eq) TTN cis:trans^(a) % ee cis^(b) % ee trans^(c) Catalase O-Tyr 0.16 0 — — — CPO^(d) S-Cys 0.40 0 — — — HRP N-His 1.00 0 — — — cyt c N-His, S-Met 1.00 12  8:92  8 −3  Mb N-His 1.00 0.8 11:89 −2 8 P450_(BM3) S-Cys 0.20 0 0 — — Hemin — 0.20 4 11:89 −1 3 ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,R) - (S,S). ^(d)Bioconversion conducted at 0.1 M citrate buffer pH = 4.0.

TABLE 9 Heme catalysts under aerobic conditions with Na₂S₂O₄.

Cat. loading Catalyst Axial ligand (% mol eq) TTN cis:trans^(a) % ee cis^(b) % ee trans^(c) Catalse O-Tyr 0.16 0 — — — CPO^(d) S-Cys 0.40 0 — — — HRP N-His 1.00 1 12:88  −3 −7 cyt c N-His, S-Met 1.00 3 9:91 −6 16 Mb N-His 1.00 6 7:93 −13 12 P450_(BM3) S-Cys 0.20 1 13:87  −38 −8 Hemin — 0.20 6 8:92 −5 1 ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,R) - (S,S). ^(d)Bioconversion conducted at 0.1 M citrate buffer pH = 4.0.

TABLE 10 Heme catalysts under aerobic conditions without Na₂S₂O₄.

Cat. loading Catalyst Axial ligand (% mol eq) TTN cis:trans^(a) % ee cis^(b) % ee trans^(c) Catalase O-Tyr 0.16 0 — — — CPO^(d) S-Cys 0.40 0 — — — HRP N-His 1.00 0 — — — cyt c N-His, S-Met 1.00 0 — — — Mb N-His 1.00 0 — — — P450_(BM3) S-Cys 0.20 0.4 46:54 −46 36 Hemin — 0.20 0 — — — ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,R) - (S,S). ^(d)Bioconversion conducted at 0.1 M citrate buffer pH = 4.0.

TABLE 11 Stereoselective P450_(BM3) cyclopropanases. Reactions were run in phosphate buffer (pH 8.0) at room temperature under argon with 30 mM styrene, 10 mM EDA, 0.2 mole % catalyst (with respect to EDA), and 10 mM sodium dithionite. Yields, diastereomeric ratios and enantiomeric excess were determined by GC analysis. Catalyst % yield^(a) TTN cis:trans % ee_(cis) ^(b) % ee_(trans) ^(c) Hemin 15 73  6:94 −1 0 P450_(BM3) 1 5 37:63 −27 −2 P450_(BM3)-F87A 1 6 38:62 26 −6 P450_(BM3)-T268A 65 323  1:99 −15 −96 H2-5-F10 59 294 16:84 −41 −63 H2A10 33 167 60:40 −95 −78 H2-4-D4 41 206 53:47 −79 −33 C3C 40 199 71:29 −94 −91 C3C-I263A 38 190 19:81 −62 −91 C3C-A328G 37 186 83:17 52 −45 C3C-T438S 59 293 92:8  −97 −66 ^(a)Based on EDA. ^(b)(R,S)-(S,R). ^(c)(R,R)-(S,S). Variant 9-10A-TS-F87V-T268A is denoted as C3C. Other sequence identities are described in Table 12.

Whether the activity and selectivity of heme-catalyzed cyclopropanation could be enhanced by engineering the protein sequence was determined. P450_(BM3) is a well-studied, soluble, self-sufficient (heme and diflavin reductase domains are fused in a single polypeptide), long-chain fatty acid monooxygenase. More than a decade of protein engineering attests to the functional plasticity of this biocatalyst (C. J. C. Whitehouse et al., Chem. Soc. Rev. 41, 1218 (2012)). Thousands of variants that exhibit monooxygenase activity on a wide range of substrates have been accumulated from using directed evolution to engineer cytochrome P450_(BM3) for synthetic applications (J. C. Lewis et al., Chimia 63, 309 (2009)). Some of these variants were tested by chiral gas chromatography for altered cyclopropanation diastero- and enantioselectivity. A panel of 92 P450_(BM3) variants, chosen for diversity of activity and protein sequence, was screened in E. coli lysate for the reaction of styrene and EDA under aerobic conditions in the presence of sodium dithionite (Tables 12 and 13). The ten most promising hits were selected for purification and subsequent characterization under standardized anaerobic reaction conditions (Tables 11 and 14).

TABLE 12 Raw data from P450_(BM3) compilation plate screen. Diastereo- and enantioselectivity were determined by gas chromatography using a chiral β-CDX column as the stationary phase. Mutations compared to wild- Absolute P450_(BM3) variants type P450_(BM3) (SEQ ID NO: 1) activity^(a) de^(b) ee (cis)^(c) CYP102A3 N/A 0.004053 −74 −8 CYP102A2 N/A 0.002963 −76 −36 CYP102A1 None 0.002240 −81 7 (P450_(BM3)) WT F87A F87A 0.001704 −28 57 WT T88L T88L 0.004522 −78 23 WT A328V A328V 0.000830 −100 N/A J⁴ V78A, T175I, A184V, F205C, 0.001334 −100 N/A S226R, H236Q, E252G, R255S, A290V, L353V 139-3⁵ V78A, H138Y, T175I, V178I, 0.001386 −86 0 A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V 9-10A⁴ R47C, V78A, K94I, P142S, 0.004292 −74 −20 T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V 9-10A L75W¹ 9-10A L75W 0.005191 −83 −8 9-10A L75I¹ 9-10A L75I 0.002267 −85 −3 9-10A A78F¹ 9-10A L78F 0.002008 −82 −35 9-10A A78S¹ 9-10A A78S 0.005098 −81 −6 9-10A A82G¹ 9-10A A82G 0.002245 −76 −7 9-10A A82F¹ 9-10A A82F #VALUE! N/A N/A 9-10A A82C¹ 9-10A A82C 0.002487 −74 16 9-10A A82I¹ 9-10A A82I 0.001031 −100 N/A 9-10A A82S¹ 9-10A A82S 0.001483 −82 14 9-10A A82L⁴ 9-10A A82L 0.000591 −100 N/A 9-10A F87A 9-10A F87A 0.001701 −61 −10 9-10A F87V¹ 9-10A F87V 0.000000 N/A N/A 9-10A F87I¹ 9-10A F87I 0.000983 −100 N/A 9-10A F87L¹ 9-10A F87L 0.000710 −100 N/A 9-10A T88C¹ 9-10A T88C 0.002516 −77 3 9-10A T260S¹ 9-10A T260S 0.004259 −82 −6 9-10A T260N¹ 9-10A T260N 0.003882 −77 15 9-10A T260L¹ 9-10A T260L 0.006173 −77 −2 9-10A A328V¹ 9-10A A328V 0.006471 −68 −8 9-10A A328M¹ 9-10A A328M 0.005180 −82 6 9-10A A328F¹ 9-10A A328F 0.002009 −63 −32 49-1A R47C, V78T, A82G, F87V, 0.001874 −75 −32 K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328L, L353V 35-7F R47C, V78F, A82S, K94I, 0.004514 −73 −52 P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328L, L353V 53-5H¹ 9-10A A78F, A82S, A328F 0.002840 −80 2 7-11D 9-10A A82F, A328V 0.036840 −24 −28 49-9B R47C, V78A, A82G, F87V, 0.000000 N/A N/A K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328L, L353V 41-5B R47C, V78F, A82G, K94I, 0.008391 −77 −17 P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328V, L353V 13-7C¹ 9-10A A78T, A328L 0.005493 −73 −43 12-10C R47C, V78A, A82G, F87V, 0.004566 −73 −21 K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328V, L353V 77-9H¹ 9-10A A78T, A82G, A328L 0.003053 −73 −34 11-8E R47C, V78A, F87V, K94I, 0.001453 −77 15 P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328L, L353V 1-12G⁴ 9-10A A82L, A328V 0.003884 −70 −19 29-3E R47C, V78A, A82F, K94I, 0.003425 −80 15 P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328F, L353V 29-10E R47C, V78F, A82G, K94I, 0.001935 −70 16 P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328F, L353V 68-8F¹ 9-10A A78F, A82G, A328L 0.004127 −72 −32 35E11⁶ R47C, V78F, A82S, K94I, 0.003600 −71 −14 P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328F, L353V, E464G, I710T 19A12⁶ 35E11 L52I, L188P, I366V 0.006909 −70 −27 ETS8⁶ 35E11 L52I, I366V 0.003966 −79 −19 (11-3)⁶ 35E11 L52I, A74S, L188P, 0.005633 −76 −39 I366V (7-7)⁶ 35E11 L52I, A74E, S82G, 0.010499 −77 −9 A184V, L188P, 1366V H2A10 9-10A TS F87V, L75A, 0.066422 −8 −94 L181A, T268A SL2-6F8 R47C, L52I, V78F, A82S, 0.000778 −100 N/A K94I, P142S,T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328L, K349N, L353V, I366V, E464G, I710T A12SL-17-4 R47C, L52I, A74E, V78F, 0.010935 −80 6 A82S, K94I, P142S, T175I, A184V, L188P, F205C, S226R, H236Q, E252G, R255S, A290V, A328F, L353V, I366V, E464G, I710T H2-2-A1² 9-10A TS F87V, L75A, 0.003042 −75 −11 L181A, L437A A12RM-2-8 R47C, L52I, A74E, V78F, 0.007705 −77 −13 A82S, K94I, P142S, T175I, A184S, L188P, F205C, S226R, H236Q, E252G, R255S, A290V, A328F, L353V, I366V, E464G, I710T H2-5-F10 9-10A TS F87V, L75A, 0.141237 −46 −56 I263A, T268A, L437A 13C9R1 L52I, I58V, L75R, F87A, 0.001980 −100 N/A H100R, S106R, F107L, A135S, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, V446I 22A3 13C9R1 F162I E434K K442E 0.004053 −70 4 I446V 2C6³ 9-10A A78L, F87A, V184T, 0.004257 −78 −15 G315S, A330V 9C7³ 9-10A C47R, A78L, F87G, 0.007258 −79 −5 I94K, A180V, V184T, G315S, A330V, Y345C B1³ 9-10A C47R, A78L, F87A, 0.002246 −61 −14 I94K, V184T, I263M, G315S, A330V B1SYN³ 9-10A C47S, N70Y, A78L, 0.002705 −76 −23 F87A, I174N, I94K, V184T, I263M, G315S, A330V H2-4-D4 9-10A TS F87V, L75A, 0.052439 57 −84 M177A, L181A, T268A, L437A E12 A87V³ 9-10A C47R, A78L, F87V, 0.001990 −65 −52 I94K, A111V, V141I, A180V, V184T, G315S, A330V GlcA4 T180A 9-10A C47R, F81W, A82S, 0.004925 −78 12 F87A, I94K H2-8-C7² 9-10A TS F87V, L75A, 0.000808 −100 N/A L181A CH-F8 9-10A L51A, C47A, F87V, 0.001126 −100 N/A I94K, L181A, C205F, S254R, I366V, L437A, E442K H2-4-H5² 9-10A TS F87V, L75A, 0.001229 −100 N/A M177A, L181A SA9 9-10A C47R, F81W, A82I, 0.004170 −81 11 F87A, I94K, A180T, A197V ManA10 9-10A C47R, F81S, A82V, 0.006340 −82 14 F87A, I94K, A180T, A197V Man1 9-10A C47R, F81L, A82T, 0.003053 −73 21 F87A, I94K MB2 9-10A C47R, F81W, A82I, 0.003282 −77 10 F87A, I94K HA62 9-10A C47R, F81A, A82L, 0.003375 −81 −5 F87A, I94K 9-10A TS V78A, P142S, T175I, A184V, 0.001920 −75 −54 S226R, H236Q, E252G, A290V, L353V, I366V, E442K 9-10A TS F87A 9-10A TS F87A 0.001546 −60 5 25F7 9-10A C47R, A74F, A78S, 0.001829 −81 43 F87A, I282K, C205F, S255R 24C4 9-10A C47R, A74I, A78L, 0.000783 −100 N/A F87A, I94K, C205F, S255R 5A1 9-10A M30T, C47R, A74F, 0.002471 −80 15 A78S, I94K, C205F, S255R, Q310L, I366V, E442K 8B3 9-10A M30T, C47R, A74F, 0.001315 −100 N/A A78S, I94K, C205F, C255R, L310Q, Q323L, I366V, N381K, R398H, E441K Determined by GC analysis on a chiral β-CDX column. ^(a)Reported as the sum of the area of the cyclopropane peaks over the area of the internal standard. ^(b)Diastereomeric excess = ([cis] − [trans])/([cis] + [trans]). ^(c)(R,S)-(S,R). ¹P. Meinhold et al., Adv. Synth. Catal. 348, 763 (2006). ²J. C. Lewis et al., Chembiochem: a European journal of chemical biology 11, 2502 (2010). ³J. C. Lewis et al., Proceedings of the National Academy of Sciences of the United States of America 106, 16550 (2009). ⁴M. W. Peters et al., J. Am. Chem. Soc. 125, 13442 (2003). ⁵A. Glieder et al., Nat. Biotechnol. 20, 1135 (2002). ⁶R. Fasan et al., Angew. Chem., Int. Ed. 46, 8414 (2007).

TABLE 13 Raw GC screening data for the chimeric P450s in the compilation plate. Chimeric P450s (heme Absolute P450 domain block sequence) activity^(a) de^(b) ee (cis)^(c) CYP102A1 11111111 0.001704 −28 56 (P450_(BM3)) F87A¹ CYP102A2 22222222 N/A N/A N/A F88A¹ CYP102A3 33333333 N/A N/A N/A F88A¹ 5R1² 32312231 0.008625 58 19 9R1² 12112333 0.0042707 58 24 12R1² 12112333 0.0701514 32 −49 C1D11R1² 21113312 0.007138 51 9 C2B12R1² 32313233 0.005914 38 −5 C2C12R1² 21313111 0.006226 28 9 C2E6R1² 11113311 0.008731 25 6 C2G9R1² 22213132 0.007975 15 31 C3D10R1² 22132231 0.004898 −16 −2 C3E4R1² 21313311 0.007893 14 17 F3H12R1² 21333233 0.005586 −56 −17 F6D8R1² 22313233 0.008088 −76 −6 C3B5R1² 23132233 0.014722 −81 4 X7R1² 22312333 0.017305 −4 −34 ^(a)Reported as the sum of the area of the cyclopropane peaks over the area of the internal standard. ^(b)Diastereomeric excess = ([cis] − [trans])/([cis] + [trans]). ^(c)(R,S)-(S,R). ¹C. R. Otey et al., PLoS Biol. 4, 789 (2006). ²M. Landwehr et al., Chem. Biol. 14, 269 (2007). Site-directed recombination of three bacterial cytochrome P450s was performed with sequence crossover sites chosen to minimize the number of disrupted contacts within the protein structure. Seven crossover sites where chosen resulting in eight sequence blocks. The numbering refers to the identity of the parent sequence at each block. For example, “12312312” refers to a sequence containing block 1 from P450 1, block 2 from P450 2, block 3 from P450 3, etc.

TABLE 14 Stereoselective P450_(BM3) based cyclopropanases.

P450 % yield^(a) TTN cis:trans^(b) % ee cis^(c) % ee trans^(d) WT 1 5 37:63 −10 −9 WTF87A 1.2 6 37:63 26 −6 H2A10 33.4 167 60:40 −95 −78 H2-4-D4 41.2 206 53:47 −79 −33 H2-5-F10 58.8 294 16:84 −41 −63 C2C12R1 1.6 8 36:64 45 1 C3E4R1 1.6 8 43:57 51 −7 X7R1 2.4 12 33:67 23 −4 12 R1 6.2 31 17:83 9 −2 C2E6 R1 4.6 23 27:73 25 −6 C2G9 R1 48 240  9:91 10 −2 7-11D 32 160 35:65 −22 −18 ^(a)based on EDA. ^(b)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(c)(R,S) - (S,R). ^(d)(R,R) - (S,S).

Five of the ten selected P450s showed improvements in activity (>100 TTN), a comprehensive range of diastereoselectivities with cis:trans ratios varying from 9:91 to 60:40, and impressive enantioselectivities (up to 95% ee, Table 14). For example, variant H2-5-F10, which contains 16 amino acid substitutions from wild type, catalyzes 294 TTN, equivalent to ˜58% yield (with respect to EDA) under these conditions. This represents a 50-fold improvement in TTN over wild type P450_(BM3). Furthermore, mutations affect both the diastereo- and enantioselectivity of cyclopropanation: H2-5-F10 favors the trans cyclopropanation product (cis:trans 16:84) with 63% ee_(trans), while variant H2A10, which catalyzes up to 167 TTN, demonstrates reversed diastereoselectivity (cis:trans 60:40) with high enantioselectivity (95% ee_(cis)).

The variant H2A10 was used to verify the role of the enzyme in catalysis and identify optimal conditions (Table 15, FIGS. 3 and 4). Heat inactivation produced diastereo- and enantioselectivities similar to those obtained using free hemin, consistent with protein denaturation and release of the cofactor. Complete inhibition was achieved by pre-incubating the bioconversion with carbon monoxide, which irreversibly binds the reduced P450 heme, confirming that catalysis occurs at the active site. Air inhibited the cyclopropanation reaction by about 50%, showing that dioxygen and EDA compete for reduced Fe^(II). Cyclopropanation was also achieved with NADPH as the reductant, confirming that the novel activity can also be driven by the endogenous electron transport machinery of the diflavin-containing reductase domain. The presence of a reducing agent in sub-stoichiometric amounts proved essential for cyclopropanation (Table 16), implying that the active species is Fe^(II) rather than the resting state Fe^(III).

TABLE 15 Controls for P450 based cyclopropanation using variant H2A10.

Conditions TTN % inhibition cis:trans^(a) % ee cis^(b) % ee trans^(c) Complete System (CS) 101 — 70:30 −95 −78 CS-Na₂S₂O₄ + NADPH 45 −55 61:39 −87 −31 CS-Na₂S₂O₄ + NADH 38 −62 53:47 −76 −19 CS-Na₂S₂O₄ 0 −100 — — — CS-P450 0 −100 — — — CS + CO 0 −100 — — — Boiled P450 146 +45 16:84 2 −2 H2A10_(heme) 85 −16 67:33 −92 −67 CS-P450 + Hemin 16 −84 15:85 −1 −2 CS (aerobic) 43 −57 67:33 −94 −76 ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,R) - (S,S).

TABLE 16 Effect of concentration of sodium dithionite on cyclopropane yield.

[Na₂S₂O₄]/mM [cyclopropanes]/mM TTN 0 0 0 1 2.59 129 5 2.72 136 10 3.34 167 20 3.13 156 50 2.79 140 100 2.71 136

Highly active variants H2A10, H2-5-F10 and H2-4-D4 have three to five active site alanine substitutions with respect to 9-10A-TS-F87V (12 mutations from wild type), which itself shows negligible cyclopropanase activity. These variants demonstrate significant differences in TTN, diastereoselectivity, and enantioselectivity (Table 11). To better understand how protein sequence controls P450-mediated cyclopropanation, 12 new variants were constructed to assess the contributions of individual alanine mutations to catalysis and stability (Table 17). T268A is key for achieving high levels of cyclopropanation activity, and this mutation alone converts inactive 9-10A-TS-F87V into an active cyclopropanase. Variant 9-10A-TS-F87V-T268A (denoted C3C) is a competent cyclopropanase (199 TTN), displays strong preference for the cis product (cis:trans 71:29), forms both diastereomers with over 90% ee, and is as stable as wild-type type 450_(BM3). Other active site alanine mutations tune the product distribution. Notably, the addition of I263A to C3C reverses diastereoselectivity (cis:trans 19:81). The effects of similar mutations introduced in the poorly active wild type P450_(BM3) were also investigated (Table 18). Impressively, P450_(BM3)-T268A, with a single mutation, is an active cyclopropanase (323 TTN, Table 11) with exquisite trans-selectivity (cis:trans 1:99) and high enantioselectivity for the major diastereomer (−96% ee_(trans), FIG. 2). Whereas C3C is a cis-selective cyclopropanase, identical active site mutations in wild type P450_(BM3) result in a trans-selective enzyme (Table 18), demonstrating that mutations outside of the active site can also influence the stereochemical outcome.

TABLE 17 Mutational analysis of alanine substitutions on 9-10A TS F87V.

Mutations relative to 9-10A TS F87V cis: % ee % ee P450 (Holo) L75A M177A L181A I263A T268A L437A TTN trans^(a) cis^(b) trans^(c) T₅₀ (° C.) 9-10A TS F87V No No No No No No 7 35:65 −41 −8 59.5 9-10A TS F87V Yes No No No No No 5 42:58 −59 −11 52.3 L75A 9-10ATS F87V No No Yes No No No 5 41:59 −27 −7 53.3 L181A 9-10A TS F87V No No No Yes No No 8 29:71 −31 −39 55.4 I263A 9-10A TS F87V No No No No Yes No 199 71:29 −94 −91 55.2 T268A (C3C) C3C I263A No No No Yes Yes No 190 19:81 −62 −91 54.0 C3C L181A No No Yes No Yes No 159 56:44 −92 −94 50.8 H2A10 Yes No Yes No Yes No 167 60:40 −95 −78 48.9 C3C L181A No No Yes Yes Yes No 203 14:86 −46 −95 50.9 I263A C3C L181A No No Yes No Yes Yes 180 27:73 −74 −98 48.4 L437A C3C L181A No No Yes Yes Yes Yes 218 9:91 −55 −96 48.2 I263A L437A 4H5 Yes Yes Yes No No No 7 32:68 −9 0 49.4 C3C I263A No No No Yes Yes Yes 267 16:84 −59 −89 50.4 L437A H2-5-F10 Yes No No Yes Yes Yes 294 16:84 −41 −63 47.5 H2-4-D4 Yes Yes Yes No Yes Yes 206 53:47 −79 −33 46.4 ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,S) - (S,S).

TABLE 18 Introducing variant 9-10A-TS-F87V-T268A related active site mutations in wild-type P450_(BM3).

Mutations relative to wild-type P450_(BM3) P450 (Holo) V78 F87 T268 I263 TTN cis:trans^(a) % ee cis^(b) % ee trans^(c) T₅₀ (° C.) WT — — — — 5 37:63 −10 −9 56.0 WT-F87A — A — — 6 38:62 26 −6 53.0 WT-F87V — V — — 9 30:70 −33 −26 52.9 WT-T268A — — A — 323  1:99 −15 −96 53.6 WT-F87V/T268A — V A — 274 32:68 −77 −99 52.0 WT- A V A — 190 32:68 −70 −20 50.8 V78A/F87V/T268 A WT- — V A A 246  7:93 8 −94 50.0 F87V/I263A/T268 A ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,R) - (S,S).

Since the design of cis-selective small-molecule catalysts for diazocarbonyl-mediated cyclopropanations has proven more challenging than their trans counterparts (A. Caballero et al., European Journal of Inorganic Chemistry, 1137 (2009)), whether further active site engineering of P450_(BM3) could provide robust cis-selective water-compatible catalysts to complement existing organometallic systems was investigated. Five active site residues (L181, I263, A328, L437, T438) were chosen for individual site-saturation mutagenesis (see, Materials and Methods). Substitutions A328G, T438A, T438S and T438P all afforded enhanced cis-selectivity (Table 19). Notably A328G also reversed the enantioselectivity for the cis-diastereomer (Table 11). C3C-T438S displayed the highest diastereo- and enantioselectivities (cis:trans 92:8 and −97% ee_(cis)) and maintained activity comparable to C3C (Table 11).

TABLE 19 Cyclopropanation activity of selected C3C_(heme) active site variants.

P450_(heme) Yield (%)^(a) TTN cis:trans^(b) % ee cis^(c) % ee trans^(d) 9-10A TS 57 286 71:29 −92 −88 F87V T268A (C3C) C3C-L181G 47 234 59:41 −89 −90 C3C-A328G 37 186 83:17 52 −45 C3C-L437F 53 265 53:47 −82 −85 C3C-L437Q 30 148 53:47 −73 −87 C3C-L437G 58 290 54:46 −88 −91 C3C-L437A 39 194 38:62 −84 −11 C3-T438A 54 273 91:9  −92 −75 C3C-T438G 15  78 73:27 −87 −59 C3C-T438S 59 293 92:8  −97 −66 C3C-T438Q 41 206 38:62 67 70 C3C-T438P 32 161 90:10 −91 −50 ^(a)Based on ED. ^(b)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(c)(R,S) - (S,R). ^(d)(R,R) - (S,S). Variant 9-10A-TS-F87V-T268A is denoted as C3C.

C3C exhibits Michaelis-Menten kinetics (FIG. 5 and Table 20) with similar K_(M) values for the olefin (˜1.5 mM) and the diazocarbene (˜5 mM). The relatively high K_(M) values reflect the lack of evolutionary pressure for these enzymes to bind these substrates. C3C exhibits a notable k_(cat) for cyclopropanation of 100 min⁻¹, comparable to the k_(cat) of many native P450s for hydroxylation, but about fifty times slower than P450_(BM3)-catalyzed fatty acid hydroxylation (Table 21). When used at 0.1 mol % equivalent, C3C-catalyzed cyclopropanations reached completion after 30 minutes. Adding more EDA equivalents leads to enhanced turnovers for cyclopropanes, with preserved C3C stereoselectivity (Table 22), confirming catalyst integrity and implying that the reaction stops because of EDA depletion rather than due to mechanistic inactivation.

TABLE 20 Michaelis-Menten parameters for P450 cyclopropanases variant 9-10A-TS-F87V-T268A (herein called C3C). k_(cat)/ k_(cat)/ k_(cat)/(K_(M-EDA×) k_(cat) K_(M-EDA) K_(M-styrene) K_(M-EDA) K_(M-styrene) K_(M-styrene)) catalyst (min⁻¹) (mM) (mM) (s⁻¹ M⁻¹) (s⁻¹ M⁻¹) (s⁻¹ M⁻¹ M⁻¹) C3C_(heme) 100 ± 24 5.2 ± 3.5 1.4 ± 0.5 320 1,100 2.1 × 10⁵

TABLE 21 Kinetic parameters for wild-type cytochrome P450s acting on their native substrates and for an engineered variant of P450_(BM3) (propane monooxygenase, PMO) acting on the non-native substrate propane. k_(cat)/K_(M-EDA) P450 Substrate k_(cat) (min⁻¹) K_(M-EDA) (mM) (s⁻¹ M⁻¹) CYP153A6¹ Octane 75 0.32 3,900 P450_(BM3) ² Lauric 5140 0.29 3.0 × 10⁵ acid PMO³ Propane 450 0.17 4.4 × 10⁴ ¹M. M. Chen et al., Advanced Synthesis & Catalysis 354, 964 (2012). ²M. A. Noble et al., Biochemical Journal 339 (Pt 2), 371 (1999). ³R. Fasan et al.,. J. Mol. Biol. 383, 1069 (2008).

TABLE 22 Effect of EDA addition at t = 30 min on variant 9-10A-TS-F87V-T268A-catalyzed cyclopropanations.

% ee Conditions TTN cis:trans^(a) cis^(b) % ee trans^(c) 10 mM EDA added at t = 0 273 ± 2.5 72:28 −92 −90 10 mM EDA added at t = 0 + 425 ± 17  73:27 −93 −89 10 mM EDA at t = 30 min TTN values are reported as the mean of triplicates ± standard deviation. ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) - (S,R). ^(c)(R,R) - (S,S).

To assess substrate scope of P450_(BM3)-catalyzed cyclopropanation, the activities of seven variants against various olefins and diazo compounds were investigated (Tables 23-27). P450 cyclopropanation is robust to both electron-donating (p-vinylanisole, p-vinyltoluene) and electron-withdrawing (p-trifluoromethylstyrene) substitutions on styrene, and 7-11D demonstrated consistent cis-selectivity for these substrates. The P450s were also active on 1,1-disubstituted olefins (e.g., α-methyl styrene), with chimeric P450 C2G9R1 forming cyclopropanes in 77% yield (with respect to EDA). The P450s were only moderately active with t-butyl diazoacetate as substrate (<30 TTN), forming the trans product with >87% selectivity and offering no advantage over free hemin (Table 27). However, for reactions involving EDA and aryl-substituted olefins, the P450s consistently outperformed the free cofactor in both activity and stereoselectivity.

TABLE 23 Substrate scope of P450 cyclopropanases: p-methylstyrene + EDA.

P450 % yield TTN cis:trans % ee cis % ee trans^(a) 7-11D 21 104 54:46 0.3 N/A H2-5-F10 44 222 11:89 14.9 N/A C2G9 R1 18 92 10:90 8.9 N/A H2A10 10 50 43:57 −84.3 N/A 9-10A TS F87V 46 228 78:22 −81.4 N/A T268A (C3C) Hemin  7 37  6:94 −1.6 N/A GC (cyclosil-B column 30 m × 0.32 mm, 0.25 μm film): oven temperature = 100° C. for 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution times: cis-cyclopropanes (21.03 and 21.18 min), trans-cyclopropanes (22.71 min). ^(a)trans-enantiomers did not resolve.

TABLE 24 Substrate scope of P450 cyclopropanases: p-vinylanisole + EDA.

P450 % yield TTN cis:trans % ee cis % ee trans^(a) 7-11D 59 297 70:30 −27 N/A H2-5-F10 73 364 11:89 38 N/A C2G9 R1 39 196 10:90 −1 N/A H2A10 16  80 40:60 −75 N/A 9-10A TS F87V 43 214 48:52 −44 N/A T268A (C3C) Hemin 19  96  7:93 0 N/A GC oven temperature = 110° C. for 8 min, 2° C./min to 180° C. then 180° C. for 30 min, 175 kPa. Cyclosil-B column (30 m × 0.25 mm, 0.25 μm film). Elution times: cis-cyclopropanes (38.74 and 39.52 min), trans-cyclopropanes (43.07 min). ^(a)Baseline resolution could not be achieved for the trans-enantiomers.

TABLE 25 Substrate scope of P450 cyclopropanases: p-(trifluoromethyl)styrene.

P450 % yield^(a) TTN^(a) cis:trans % ee cis % ee trans 7-11D 24 120 76:24 31 59 H2-5-F10 40 198 26:74 72 −65 C2G9 R1 18 89 10:90 4 0 H2A10 9 47 26:74 −24 22 9-10A TS F87V 42 211 39:61 54 −93 T268A (C3C) Hemin 2 9 11:89 1 1 ^(a)Assumed the same detector response factor as for ethyl 2-(4-methylphenyl)cyclopropane-1-carboxylate. GC (cyclosil-B column 30 m × 0.25 mm, 0.25 μm film): oven temperature = 110° C. for 8 min, 2° C./min to 180° C. for 30 min, 175 kPa. Elution times: cis-cyclopropanes (27.26 and 28.11 min), trans-cyclopropanes (30.78 and 30.99 min).

TABLE 26 Substrate scope of P450 cyclopropanases: α-methyl styrene.

P450 % yield TTN Z:E % ee (Z) % ee (E)^(a) 7-11D 31 157 41:49 42 N/A H2-5-F10 66 329 21:79 −14 N/A C2G9 R1 77 387 16:84 −4 N/A H2A10 34 168 19:81 −31 N/A 9-10A TS F87V 26 127 16:84 −6 N/A T268A (C3C) WT F87V T268A 62 312  7:93 3 N/A Hemin 15  77 24:76 0 N/A GC oven temperature = 100° C. for 5 min, 1° C./min up to 135° C., 135° C. for 10 min, 10° C./min up to 200° C., 200° C. for 5 min. Cyclosil-B column (30 m × 0.32 mm, 0.25 μm film). Elution times: Z-cyclopropanes (34.96 and 35.33 min), E-cyclopropanes (39.34 and 39.61 min). ^(a)trans-enantiomers did not separate to baseline resolution.

TABLE 27 Substrate scope of P450 cyclopropanases: t-butyl diazoacetate.

P450 % yield^(a) TTN^(a) cis:trans WT F87V T268A 1.4 7 4:96 7-11D 11 54 13:87  H2-5-F10 18 90 3:97 H2A10 24 120 3:97 9-10A TS F87V 0.3 2 3:97 T268A (C3C) Hemin 20 100 4:96 ^(a)Assumed the same detector response factor as for ethyl 2-(4-methylphenyl)cyclopropane-1-carboxylate. GC (cyclosil-B column 30 m × 0.32 mm, 0.25 μm film): oven temperature = 100° C. for 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution times: cis-cyclopropanes (21.66 min), trans-cyclopropanes (23.31 min). Cis- and trans-enantiomers did not resolve.

Designing enzymes that catalyze reactions not observed in nature constitutes a contemporary challenge in protein engineering (J. B. Siegel et al., Science 329, 309 (2010)). Working from a natural enzyme with promiscuous reactivity, this example demonstrates the construction of a cyclopropanase that exhibits kinetics comparable to natural enzymes, albeit with pre-activated reagents. Discovering catalysts for non-natural bond-disconnections by screening natural enzymes against synthetic reagents chosen based on chemical intuition offers a simple strategy for identifying enzymes with basal levels of novel activity. As shown herein, a single mutation can be enough to promote the new activity and achieve synthetically useful stereoselectivities. The established reaction promiscuity of natural enzymes (U. T. Bornscheuer et al., Angew. Chem. Int. Ed. 43, 6032 (2004)) and the ease with which cyclopropanase activity could be installed into P450_(BM3) indicates that this approach will be useful for other synthetically important transformations for which biological counterparts do not yet exist.

Materials and Methods

Unless otherwise noted, all chemicals and reagents for chemical reactions were obtained from commercial suppliers (Sigmα-Aldrich, Acros) and used without further purification. The following heme proteins were all purchased from Sigmα-Aldrich: myoglobin (from equine heart), peroxidase II (from horseradish), cytochrome c (from bovine heart), catalase (from Corynebacterium glutamicum) and chloroperoxidase (from Caldariomyces fumago). Silica gel chromatography purifications were carried out using AMD Silica Gel 60, 230-400 mesh. ¹H and ¹³C NMR spectra were recorded on either a Varian Mercury 300 spectrometer (300 MHz and 75 MHz, respectively), or a Varian Inova 500 MHz (500 MHz and 125 MHz, respectively), and are internally referenced to residual solvent peak. Data for ¹H NMR are reported in the conventional form: chemical shift (δ ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling constant (Hz), integration. Data for ¹³C are reported in terms of chemical shift (δ ppm) and multiplicity. High-resolution mass spectra were obtained with a JEOL JMS-600H High Resolution Mass Spectrometer at the California Institute of Technology Mass Spectral Facility. Reactions were monitored using thin layer chromatography (Merck 60 silica gel plates) using an UV-lamp for visualization. Optical rotation was measured using a JASCO P-2000 Polarimeter.

Gas chromatography (GC) analyses were carried out using a Shimadzu GC-17A gas chromatograph, a FID detector, and J&W scientific cyclosil-B columns (30 m×0.32 mm, 0.25 μM film and 30 m×0.25 mm, 0.25 μm film). High-performance liquid chromatography (HPLC) was carried out using an Agilent 1200 series, an UV detector, and an Agilent XDB-C18 column (4.6×150 mm, 5 μm). Cyclopropane product standards for the reaction of ethyl diazoacetate (EDA) with styrene (ethyl 2-phenylcyclopropane-1-carboxylate) and α-methylstyrene (ethyl 2-methyl-2-pehnylcyclopropane-1-carboxylate) were prepared as reported (A. Penoni et al., European Journal of Inorganic Chemistry, 1452 (2003)). These standards were used in co-injection experiments to determine the authenticity of enzyme-catalyzed cyclopropanes. Authentic P450 catalyzed cyclopropane samples were also prepared as described below and were characterized by NMR (¹H and ¹³C) and mass spectrometry. Azides 5 and 8, and benzosultam standards 6 and 9 were prepared as reported (J. V. Ruppel et al., Org. Lett. 9, 4889 (2007)). Benzosultam 11 was purchased from Sigma. These standards were used in co-injection experiments to determine the authenticity of P450-catalyzed benzosultams. Authentic P450 catalyzed benzosultam samples were also prepared as described below and were characterized by NMR (¹H and ¹³C) and mass spectrometry.

Plasmids pCWori[BM3] and pET22 were used as cloning vectors. Site-directed mutagenesis was accomplished by standard overlap mutagenesis using primers baring desired mutations (IDT, San Diego, Calif.). Electro-competent Escherichia coli cells were prepared following the protocol of Sambrook et al., Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, New York, 1989), vol. 2. Restriction enzymes BamHI, EcoRI, XhoI, Phusion polymerase, and T4 ligase were purchased from New England Biolabs (NEB, Ipswich, Mass.). Alkaline phosphatase was obtained from Roche (Nutley, N.J.). The 1,000× trace metal mix used in expression cultures contained: 50 mM FeCl₃, 20 mM CaCl₂, 10 mM MnSO₄, 10 mM ZnSO₄, 2 mM CoSO₄, 2 mM CuCl₂, 2 mM NiCl₂, 2 mM Na₂MoO₄, and 2 mM H₃BO₃.

Enzyme Library Screening.

Libraries are stored at −78° C. as glycerol stocks (Luriα-Bertani medium (LB_(amp)), 150 μL, 25% v/v glycerol with 0.1 mg/mL ampicillin) in 96-well plates. These stocks were used to inoculate 96-shallow-well plates containing 300 μL LB_(amp) medium using a 96-pin stamp. Single colonies from site saturation libraries were picked with toothpicks and used to inoculate 300 μL of LB_(amp). The cells were incubated at 37° C., 250 rpm, and 80% relative humidity overnight. After 16 h, 50 μL aliquots of these over night cultures were transferred into 2 mL, deep-well plates containing terrific broth (TB_(amp)) (800 μL containing 0.1 mg/mL ampicillin, 1 μL/mL trace metal mix and 20 mg L⁻¹ aminolevulinic acid) using a Multimek 96-channel pipetting robot (Beckman Coulter, Fullerton, Calif.). The cultures were incubated at 37° C. for 3 h and 30 min, and 30 min after reducing the incubation temperature to 25° C. (250 rpm, 80% relative humidity), 50 μL isopropyl β-D-1-thiogalactopyranoside (IPTG, 4.5 mM in TB_(amp)) was added, and the cultures were allowed to continue for another 24 h at 25° C. (250 rpm, 80% relative humidity). Cells were then pelleted (3,000×g, 15 min, 4° C.) and stored at −20° C. until further use, but at least for 2 h. For cell lysis, plates were allowed to thaw for 30 min at room temperature and then cell pellets were resuspended in 275 μL phosphate buffer (0.1 M, pH=8.0, 0.65 mg/mL lysozyme, 10 mM magnesium chloride and 40 U/mL DNAse I). The lysing cells were incubated at 37° C. for 1 h. Cell debris was separated by centrifugation at 5,000×g and 4° C. for 15 min. The resulting crude lysates were then transferred to 96-well microtiter plates for CO assays and to 2 mL deep well plates for bioconversions.

CO Binding Assay.

P450_(BM3) variants in cell lysate (40 μL) were diluted with 60 μL phosphate buffer (0.1 M, pH=8.0). To this solution was added 160 μL sodium dithionite (0.1 M in phosphate buffer, 0.1 M, pH=8.0). The absorbance at 450 and 490 nm was recorded using a Tecan M1000 UV/Vis plate reader, and the microtiter plates were placed in a vacuum chamber. The chamber was sealed, evacuated to approximately −15 in Hg, purged with CO gas, and incubated for 30 min. The plates were then removed and the absorbance at 450 and 490 nm was again recorded using a plate reader. The difference spectra could then be used to determine the P450 concentration in each well as previously described (C. R. Otey, in Methods in Molecular Biology: Directed Enzyme Evolution, F. H. Arnold, G. Georgiou, Eds. (Humana Press, Totowa, N.J., 2003), vol. 230).

P450 Expression and Purification.

For the enzymatic transformations, P450_(BM3) variants were used in purified form. Enzyme batches were prepared as follows. One liter TB_(amp) was inoculated with an overnight culture (100 mL, LB_(amp)) of recombinant E. coli DHSa cells harboring a pCWori plasmid encoding the P450 variant under the control of the tac promoter. After 3.5 h of incubation at 37° C. and 250 rpm shaking (OD₆₀₀ ca. 1.8), the incubation temperature was reduced to 25° C. (30 min), and the cultures were induced by adding IPTG to a final concentration of 0.5 mM. The cultures were allowed to continue for another 24 hours at this temperature. After harvesting the cells by centrifugation (4° C., 15 min, 3,000×g), the cell pellet was stored at −20° C. until further use but at least for 2 h. The cell pellet was resuspended in 25 mM Tris.HCl buffer (pH 7.5 at 25° C.) and cells were lysed by sonication (2×1 min, output control 5, 50% duty cycle; Sonicator, Heat Systems—Ultrasonic, Inc.). Cell debris was removed by centrifugation for 20 min at 4° C. and 27,000×g and the supernatant was subjected to anion exchange chromatography on a Q Sepharose column (HiTrap™ Q HP, GE Healthcare, Piscataway, N.J.) using an AKTAxpress purifier FPLC system (GE healthcare). The P450 was eluted from the Q column by running a gradient from 0 to 0.5 M NaCl over 10 column volumes (P450 elutes at 0.35 M NaCl). The P450 fractions were collected and concentrated using a 30 kDa molecular weight cut-off centrifugal filter and buffer-exchanged with 0.1 M phosphate buffer (pH=8.0). The purified protein was flash-frozen on dry ice and stored at −20° C. P450 concentration was determined in triplicate using the CO binding assay described above (10 μL P450 and 190 μL 0.1 M phosphate buffer, pH 8.0, per well).

Thermostability Measurements.

Duplicate measurements were taken for all values reported on Tables 17 and 18. Purified P450 solutions (4 μM, 200 μL) were heated in a thermocycler (Eppendorf) over a range of temperatures (38° C.-65° C.) for 10 min followed by rapid cooling to 4° C. for 1 min. The precipitate was removed by centrifugation. The concentration of folded P450 remaining in the supernatant was measured by CO-difference spectroscopy (as described above). The temperature at which half of the protein was denatured (T₅₀) was determined by fitting the data to the equation: f(T)=100/(1+exp(a*(T−T₅₀))).

Typical Procedure for Small-Scale Cyclopropanation Bioconversions Under Anaerobic Conditions.

Small-scale reactions (400 L) were conducted in 2 mL crimp vials (Agilent Technologies, San Diego, Calif.). P450 solution (80 μL, 100 μM) was added to the vial with a small stir bar before crimp sealing with a silicone septum. Phosphate buffer (260 μL, 0.1 M, pH=8.0) and 40 μL of a solution of the reductant (100 mM sodium dithionite, or 20 mM NADPH) were combined in a larger crimp sealed vial and degassed by bubbling argon through the solution for at least 5 min (FIG. 3). In the meantime, the headspace of the 2 mL reaction vial with the P450 solution was made anaerobic by flushing argon over the protein solution (with no bubbling). When multiple reactions were conducted in parallel, up to 8 reaction vials were degassed in series via cannula. The buffer/reductant solution (300 μL) was syringed into the reaction vial, while under argon. The gas lines were disconnected from the reaction vial before placing the vials on a plate stirrer. A 40× styrene solution in MeOH (10 μL, typically 1.2 M) was added to the reaction vial via a glass syringe, and left to stir for about 30 s. A 40×EDA solution in MeOH was then added (10 μlt, typically 400 mM) and the reaction was left stirring for the appropriate time. The final concentrations of the reagents were typically: 30 mM styrene, 10 mM EDA, 10 mM sodium dithionite, 20 μM P450.

The reaction was quenched by adding 30 μL HCl (3M) via syringe to the sealed reaction vial. The vials were opened and 20 μL internal standard (20 mM 2-phenylethanol in MeOH) was added followed by 1 mL ethyl acetate. This mixture was transferred to a 1.8 mL eppendorf tube which was vortexed and centrifuged (16,000×g 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by chiral phase GC.

A slightly modified work-up was implemented for kinetic experiments. The reactions were quenched after the set time by syringing 1 mL EtOAc to the closed vials and immediately vortexing the mixture. The vials were then opened and 20 μL internal standard was added. The mixture was transferred to a 1.8 mL eppendorf tube, vortexed and centrifuged (16,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by GC.

Typical Procedure for Preparative-Scale Cyclopropanation Bioconversions Under Anaerobic Conditions.

The P450 solution was added to a Schlenk flask with a stir bar. With the flask kept on ice, the head-space was evacuated and back-filled with argon (4×) with care not to foam the protein solution. Phosphate buffer and reductant were pre-mixed and degassed together in a separate round-bottom-flask by bubbling argon through the solution for 20 min. The buffer/reductant solution was transferred to the Schlenk flask via syringe. Styrene was added under argon and left to mix for 1 min. EDA was added dropwise under argon. The solution was left to stir under argon until reaction completion. The reaction was quenched under argon by adding hydrochloric acid (3 M) to adjust the pH to 4, before opening the Schlenk flask. The reaction mixture was stirred with sodium chloride and dichloromethane (CH₂Cl₂). The combined emulsion layers were then filtered through Celite to break the emulsion and the Celite pad was rinsed with 3×20 mL CH₂Cl₂. The resulting biphasic mixture was transferred to a separating funnel and the organic phase was removed. The remaining aqueous phase was re-extracted with 3×40 mL CH₂Cl₂. The combined organic extracts were dried with sodium sulfate, filtered, and concentrated. The resulting residue was purified by SiO₂ chromatography.

Summary of Mutations (with Respect to Wild-Type P450_(BM3)) of P450 Cyclopropanases and Aminases.

-   7-11D (P. Meinhold et al., Adv. Synth. Catal. 348, 763 (2006)):     R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G,     R255S, A290V, L353V, A82F, A328V -   9-10A TS (J. C. Lewis et al., Chembiochem: A European Journal of     Chemical Biology 11, 2502 (2010)): V78A, P142S, T175I, A184V, S226R,     H236Q, E252G, A290V, L353V, I366V, E442K -   H2A10: 9-10A TS F87V, L75A, L181A, T268A -   H2-5-F10: 9-10A TS F87V, L75A, I263A, T268A, L437A -   H2-4-D4: 9-10A TS F87V, L75A, M177A, L181A, T268A, L437A -   C3C: 9-10A TS F87V T268A -   B1SYN (J. C. Lewis et al., Proceedings of the National Academy of     Sciences USA 106, 16550 (2009)): R47C, V78A, K94I, P142S, T175I,     A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, C47S, N70Y,     A78L, F87A, I174N, 194K, V184T, I263M, G315S, A330V

Supplementary Data

Preliminary Experiments with Heme Proteins

The following six heme proteins were initially screened for ‘cyclopropanase’ activity: catalase, chloroperoxidase (CPO), horseradish peroxidase (HRP), cytochrome C (cyt c), myoglobin (Mb) and P450_(BM3). Small-scale (400 μL) reactions were conducted as described in section II and were analyzed by GC (cyclosil-B 30 m×0.25 mm×0.25 μm): oven temperature=130° C. Table 7 shows heme catalysts under anaerobic conditions with sodium dithionite (Na₂S₂O₄). Table 8 shows heme catalysts under anaerobic conditions without Na₂S₂O₄. Table 9 shows heme catalysts under aerobic conditions with Na₂S₂O₄. Table 10 shows heme catalysts under aerobic conditions without Na₂S₂O₄.

Screening P450BM3 Variants for Cyclopropanation Activity

Lysate Screening Under Aerobic Conditions.

The 92 P450_(BM3) variants in the compilation plate (Table 12) represent a diverse selection of P450_(BM3) scaffolds that have previously been engineered for monooxygenase activity on a variety of substrates, including but not limited to short alkane hydroxylation, demethylation of protected monosaccharides and oxidation of lead drug compounds. These P450_(BM3) variants carry various mutations (Table 12) accumulated along sequential rounds of engineering efforts for activity towards the target substrates. The compilation plate was expressed and lysed as described in above (enzyme library screening). 150 μL lysate was transferred (Multimek 96-channel pipetting robot, Beckman Coulter, Fullerton, Calif.) to a 2 mL deep well plate, with 50 μL of 120 mM Na₂S₂O₄ in 0.1 M KPi (pH=8.0). 100 μL of a 30 mM styrene, 60 mM EDA mixed solution in 15% MeOH in 0.1 M KPi (pH=8.0) was added to the plate to initiate the reaction. The plate was sealed and was left shaking (300 rpm) for four hours. The plastic seal was removed and 30 μL HCl (3 M) was added to quench the reaction followed by 20 μl, of an internal standard solution (20 mM α-methylstyrene in methanol). The reactions were extracted by adding 500 μL EtOAc and carefully vortexing the plate. The plate was centrifuged (1,700×g) to separate the biphasic mixture. The top organic layer was transferred (2×150 μL) to a separate deep well plate. The extracts for each of the 92 reactions were dried through 92 separate anhydrous sodium sulfate plugs. The dried extracts were analyzed by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=60° C. 3 min, 7.5° C./min to 160° C., 20° C./min to 250° C., 250° C. 2 min, cis-cyclopropanes (20.3 min and 20.45 min), trans-cyclopropanes (21.8 min).

Determining the Cyclopropanation Activity of the Top 10 Hits in Tables 12-13 Under Anaerobic Conditions.

Small-scale reactions (400 μL total volume) were conducted as described above and were analyzed by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=100° C. 5 min, 1° C./min to 135° C., 135° C. 10 min, 10° C./min to 200° C., 200° C. 5 min, cis-cyclopropanes (39.40 min and 40.20 min), trans-cyclopropanes (44.69 min and 45.00 min). Table 14 shows stereoselective P450BM3 based cyclopropanases.

Experimental Characterization of P450BM3 Cyclopropanases

Controls to Confirm the Enzymatic Cyclopropanation Activity of Variant H2A10.

Small scale reactions (400 μL total volume) were set up and worked-up as described above. For the carbon monoxide (CO) inhibition experiment, the reaction vial and the buffer/reductant vial were purged with CO after having been purged with argon. For the boiled P450 experiment, a 100 μM solution of variant H2A10 was heated at 60° C. for 10 min. For the hemin experiment, hemin (80 μL) was added from a 1 mM solution in 50% DMSO-H₂O, such that its final concentration in the reaction was 200 μM. Complete System=10 mM styrene, 20 mM EDA, 20 mM Na₂S₂O₄, 20 μM P450 (H2A10) under anaerobic conditions. The dried ethyl acetate extracts were analyzed by chiral phase GC, using 2-phenylethanol as an internal standard (injector temperature=300° C., oven temperature=100° C. for 5 min, 1° C./min ramp up to 135° C., 135° C. for 10 min, 10° C./min ramp up to 200° C., 200° C. for 5 min). Elution time: cis-cyclopropanes (39.40 min and 40.20 min), trans-cyclopropanes (44.69 min and 45.00 min). Table 15 shows controls for P450 based cyclopropanation using variant H2A10.

Optimizing Cyclopropanation Reaction Conditions for Variant H2A10.

Small-scale reactions (400 μL final volume) were set up and worked up as described above. The dried ethyl acetate extracts were analyzed by chiral phase GC, using 2-phenylethanol as an internal standard (injector temperature=300° C., oven temperature=100° C. for 5 min, 5° C./min ramp up to 200° C., 20° C./min ramp up to 250° C., 250° C. for 5 min). Elution time: cis-cyclopropanes (19.20 min and 19.33 min), trans-cyclopropanes (20.44 min). The reaction conditions that gave optimal yields of cyclopropanes (with respect to EDA) were: 30 mM styrene, 10 mM EDA and 20 μM P450 and were used in subsequent experiments.

Styrene Concentration.

FIG. 3 illustrates the effect of styrene concentration on cyclopropane yield.

P450 Concentration.

FIG. 4 illustrates the effect of P450 (H2A10) concentration on cyclopropane yield.

Dithionite Concentration.

Table 16 shows the effect of the concentration of Na₂S₂O₄ on cyclopropane yield.

Mutational Analysis of Active Site Alanine Substitutions in 9-10A TS F87V.

Table 17 shows a mutational analysis of alanine substitutions on 9-10A TS F87V.

Sequential Introduction of BM3-CIS Active Site Mutations in Wild-Type P450_(BM3).

Table 18 shows introducing BM3-CIS related active site mutations in wild-type P450_(BM3).

Active Site Saturation Mutagenesis of C3C_(heme)

Library Construction.

To simplify library construction and screening, only the C3C heme domain, which comprises residues 1-462 was used. This truncated enzymes lacks the P450 native reductase and exhibits similar activity and stereochemical control to the holo enzyme using dithionite as a reductant, but not NADPH. P450 site-directed mutagenesis and site-saturation libraries were assembled from PCR fragments generated from oligonucleotides containing the desired codon mutation or a degenerate NNK (or for reverse primers, the reverse complement MNN; where N=A,T,G,C,K=G,T and M=A,C) codon, which codes for all 20 amino acids and the TAG stop codon. PCR fragments were assembled using either standard overlap extension PCR or through restriction cloning using the Type IIS restriction enzyme, BsaI, depending on convenience.

Lysate Screening Under Aerobic Conditions.

The compilation plate was expressed and lysed as described above (enzyme library screening). 150 μL lysate was transferred (Multimek 96-channel pipetting robot, Beckman Coulter, Fullerton, Calif.) to a 2 mL deep well plate, with 50 μL of 120 mM Na₂S₂O₄ in 0.1 M KPi (pH=8.0). 100 μL of a 90 mM styrene, 30 mM EDA mixed solution in 15% MeOH in 0.1 M KPi (pH=8.0) was added to the plate to initiate the reaction. The plate was sealed and was left shaking (300 rpm) for four hours. The plastic seal was removed and 30 μL HCl (3 M) was added to quench the reaction followed by 20 μL of an internal standard solution (20 mM 2-phenylethanol in methanol). Acetonitrile (400 μL) was added before carefully vortexing the plate. The plate was centrifuged (1,700×g), the supernatant was filtered (1 μm glass, 96 well filter plate, Pall) and transferred (150 μL) to a 96-well microtiter plate (Agilent). Reactions were analyzed by reverse-phase HPLC (210 nm): 50% acetonitrile-water, 1.0 mL min⁻¹, cis-cyclopropanes (7.6 min), trans-cyclopropanes (9.7 min). Hits were selected based on enhancement of cis-selectivity over parent C3C.

Determining the Cyclopropanation Activity of Hits from the Site-Saturation Libraries Under Anaerobic Conditions.

Small-scale reactions (400 μL total volume) were conducted as described above and were analyzed by GC (cyclosil-B 30 m×0.25 mm×0.25 μm): oven temperature=130° C., 175 kPa, cis-cyclopropanes (39.40 min and 40.20 min), trans-cyclopropanes (44.69 min and 45.00 min). Table 19 shows the cyclopropanation activity of selected C3C_(heme) active site variants

Kinetic Characterization of C3C

Determination of initial rates. Both styrene and EDA concentrations were varied in the presence of the P450s expressed as the heme-domain (0.5 or 1.0 μM C3C_(heme)). Reactions were set up in phosphate buffer (pH=8.0) with sodium dithionite as the reductant at 298 K, and were worked-up as described above. Three time points were taken and used to determine the rate of product formation by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=100° C. 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution time: cis-cyclopropanes (19.20 min and 19.33 min), trans-cyclopropanes (20.44 min). Kinetic parameters were determined by fitting the data to the standard Michaelis-Menten model.

FIG. 5 illustrates the initial velocities plot for C3C_(heme). (A) EDA concentration was varied at a saturating concentration of styrene (30 mM). (B) Styrene concentration was varied at a fixed concentration of EDA (20 mM). Table 20 shows the Michaelis-Menten parameters for P450 cyclopropanation catalysts. Table 21 shows kinetic parameters for wild-type cytochrome P450s acting on their native substrates and for an engineered variant of P450_(BM3) (propane monooxygenase, PMO) acting on the non-native substrate propane. Table 22 shows the effect of EDA addition at t=30 min on C3C-catalyzed cyclopropanations.

Substrate Scope of P450 Cyclopropanases

Small-Scale Reactions.

Selected P450 catalysts were surveyed at a small-scale (400 total volume) for each combination of reagents (olefins and diazo esters). The small-scale anaerobic bioconversions were conducted as described above and were analyzed by GC. Table 23 shows the substrate scope of P450 cyclopropanation catalysts: p-methylstyrene+EDA. Table 24 shows the substrate scope of P450 cyclopropanation catalysts: p-vinylanisole+EDA. Table 25 shows the substrate scope of P450 cyclopropanation catalysts: p-(trifluoromethyl)styrene. Table 26 shows the substrate scope of P450 cyclopropanation catalysts: α-methyl styrene. Table 27 shows the substrate scope of P450 cyclopropanation catalysts: t-butyl diazoacetate.

Preparative-Scale Bioconversions.

These reactions were conducted anaerobically as described above.

Cyclopropanation of Styrene with EDA.

Prepared using 1.5 mmol styrene (3 equiv), 0.5 mmol EDA (1 equiv) and 1 μmol C3C_(heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 25 mg of the cis-cyclopropane (1) and 8 mg of a mixture of cyclopropanes with trans (2) in 5:1 excess over cis (C. J. Sanders et al., Tetrahedron: Asymmetry 12, 1055 (2001); M. Lenes Rosenberg et al., Organic Letters 11, 547 (2009); Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007)). Diagnostic data for the cis-cyclopropane 1: ¹H NMR (CDCl₃, 500 MHz): δ 7.28 (m, 4H), 7.21 (m, 1H), 3.89 (q, J=7.1 Hz, 2H), 2.60 (m, 1H), 2.10 (m, 1H), 1.73 (m, 1H), 1.35 (m, 1H), 0.99 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 170.99, 136.56, 129.31, 127.88, 126.63, 60.18, 25.47, 21.80, 14.02, 11.12; [α]²⁵ _(D)=−7.056° (c 0.83, CHCl₃). Diagnostic data for the trans-cyclopropane 2: ¹H NMR (CDCl₃, 500 MHz): δ 7.20 (m, 3H), 7.03 (m, 2H), 4.10 (q, J=7.1 Hz, 2H), 2.45 (m, 1H), 1.83 (m, 1H), 1.53 (m, 1H), 1.23 (m, 1H), 1.21 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 173.43, 140.13, 128.46, 126.55, 126.16, 60.72, 26.18, 24.20, 17.09, 14.27; [α]²⁵ _(D)=+ 199.2° (c 0.50, CHCl₃). MS (E1+) m/z: 190 (M⁺), 162 (PhCH(CH₂)CHCO₂ ⁺), 145 (PhCH(CH₂)CHCO⁺). The absolute configuration of compounds 1 and 2 was determined by comparison of the sign of their optical rotations with that reported (N. Watanabe et al., Heterocycles 42, 537 (1996)). The enantiomeric excess was determined to be 92% for the cis-cyclopropane and 88% for the trans-cyclopropane by GC.

Cyclopropanation of p-Methylstyrene with EDA.

Prepared using 1.5 mmol styrene (3 equiv), 0.5 mmol EDA (1 equiv) and 1 mol_(C3C heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 10 mg of the cis-cyclopropane (3) and 16 mg of a mixture of cyclopropanes with trans(4): cis/2:1 (Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007)). Diagnostic data for the cis-cyclopropane 3: ¹H NMR (CDCl₃, 500 MHz): δ 7.17 (d, J=8.0 Hz, 2H), 7.08 (d, J=8.0 Hz, 2H), 3.91 (q, J=7.1 Hz, 2H), 2.56 (m, 1H), 2.32 (s, 3H) 2.06 (m, 1H), 1.69 (m, 1H), 1.32 (m, 1H), 1.02 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 171.12, 136.12, 133.42, 129.14, 128.60, 60.17, 25.23, 21.68, 21.10, 14.08, 11.21. Diagnostic data for the trans-cyclopropane 4: ¹H NMR (CDCl₃, 500 MHz): δ 7.09 (d, J=8.0 Hz, 2H), 7.01 (d, J=8.0 Hz, 2H), 4.19 (q, J=7.1

Hz, 2H), 2.50 (m, 1H), 2.33 (s, 3H), 1.88 (m, 1H), 1.59 (m, 1H), 1.33 (m, 1H), 1.29 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 173.58, 137.04, 136.08, 129.12, 126.10, 60.66, 25.94, 24.06, 21.11, 16.96, 14.28. MS (EI⁺) m/z: 204 (M⁺), 175 ([M-Et]⁺) 131 ([M-COOEt]⁺). The enantiomeric excess was determined to be 82% for the cis-cyclopropane by GC. Baseline resolution of the trans-enantiomers could not be achieved.

Cyclopropanation of p-Methoxystyrene with EDA.

Prepared using 1.5 mmol styrene (3 equiv), 0.5 mmol EDA (1 equiv) and 1 μmol C3C_(heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 16 mg of the trans-cyclopropane (6) and 3 mg of a mixture of cyclopropanes with cis:trans/5:1 (Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007)). Diagnostic data for the trans-cyclopropane 6: 6.96 (m, 3H), 6.75 (m, 2H), 4.09 (q, J=7.1 Hz, 2H), 3.72 (s, 3H), 2.41 (m, 1H), 1.75 (m, 1H), 1.48 (m, 1H), 1.21 (t, J=7.1 Hz, 3H), 1.18 (m, 1H). MS (EI⁺) m/z: 220 (M⁺), 191 ([M-Et]⁺), 175 ([M-EtO]⁺), 147 ([M-COOEt]⁺). The enantiomeric excess was determined to be 38% for the cis-cyclopropane by GC. The trans-enantiomers did not resolve to baseline resolution.

Cyclopropanation of Styrene with t-Butyl Diazo Acetate.

Prepared using 0.75 mmol styrene (3 equiv), 0.24 mmol t-BuDA (1 equiv) and 0.5 μmol C3C_(heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 9 mg of the trans-cyclopropane (8) (C. J. Sanders et al., Tetrahedron: Asymmetry 12, 1055 (2001); Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007)). Diagnostic data for the trans-cyclopropane 4: ¹H NMR (CDCl₃, 500 MHz): δ 7.20 (m, 2H), 7.12 (m, 1H), 7.02 (m, 2H), 2.36 (m, 1H), 1.76 (m, 1H), 1.45 (m, 1H), 1.40 (s, 9H), 1.16 (m, 1H); ¹³C NMR (CDCl₃, 125 MHz): δ 172.58, 140.52, 128.42, 126.32, 126.07, 80.57, 28.17, 25.75, 25.31, 17.08. MS (EI⁺) m/z: 218 (M⁺), 145 ([M-OtBu]⁺).

Example 2 In Vivo and In Vitro Olefin Cyclopropanation Catalyzed by Heme Enzymes

This example illustrates the use of heme containing enzymes to catalyze the conversion of olefins to various products containing one or more cyclopropane functional groups. In certain aspects, this example demonstrates novel variants of cytochrome P450_(BM3) (CYP102A1). These variants have an improved ability to use diazo esters as carbene precursors and cyclopropanate various olefins. Preferred variants include cytochrome P450_(BM3) mutants having C400S and T268A amino acid substitutions and engineered variants of other P450s having the equivalent substitutions. Variants with axial serine coordination efficiently catalyze the cyclopropanation reaction in whole cells sustaining over 10,000 total turnovers.

Introduction

The many strategies for functionalizing C═C and C—H bonds that have evolved in Nature have captivated the imaginations of chemists and form the foundation of biomimetic chemistry (J. T. Groves, Proceedings of the National Academy of Sciences U.S.A. 100, 3569 (2003); R. Breslow, Journal of Biological Chemistry 284, 1337 (2009)). The reverse of this, using inspiration from synthetic chemistry to discover and develop new biocatalysts, is a nascent frontier in molecular engineering whose recent highlights include C—H activation by artificial rhodium enzymes (T. K. Hyster et al., Science 338, 500 (2012)) and the de novo design of Diels-Alderases (T. K. Hyster et al., Science 338, 500 (2012); J. B. Siegel et al., Science 329, 309 (2010)). Synthetic chemists have developed powerful methods for direct C═C and C—H functionalization based on transition metal-catalyzed carbenoid and nitrenoid transfers, reactions that are widely used to synthesize natural product intermediates and pharmaceuticals (H. M. L. Davies et al., Nature 451, 417 (2008)). The asymmetric cyclopropanation of olefins with high-energy carbene precursors (e.g., acceptor-substituted diazo reagents) is a hallmark reaction that generates up to 3 stereogenic centers in a single step to make the important cyclopropane motif, featured in many natural products and therapeutic agents (H. Lebel et al., Chemical Reviews 103, 977 (2003)). Limited to using physiologically accessible reagents, Nature catalyzes intermolecular cyclopropane formation through wholly different strategies, typically involving olefin addition to the methyl cation of S-adenosyl methionine or through cyclization of dimethylallyl pyrophosphate-derived allylic carbenium ions (L. A. Wessjohann et al., Chemical Reviews 103, 1625 (2003)). As a result, the diverse cyclopropanation products that can be formed by metallocarbene chemistry cannot be readily accessed by engineering natural cyclopropanation enzymes. This example describes a natural metalloenzyme, the iron-heme-containing cytochrome P450, engineered to catalyze formal carbenoid transfers, thereby combining the high levels of regio- and stereoselectivity of enzymes with the synthetic versatility of carbene-based strategies. An enzyme is non-toxic, could be used in a primarily aqueous medium at ambient temperatures, and could be significantly less expensive to prepare than current transition metal catalysts.

Results

Members of the cytochrome P450 enzyme family catalyze myriad oxidative transformations, including hydroxylation, epoxidation, oxidative ring coupling, heteroatom release, and heteroatom oxygenation (E. M. Isin et al., Biochim. Biophys. Acta, 1770, 314 (2007)). Most transformations encompassed by this broad catalytic scope manifest the reactivity of the same high-valent iron-oxene intermediate (Compound I, FIG. 1). Inspired by the impressive chemo-, regio- and stereoselectivities with which cytochrome P450s can insert oxygen atoms into C—H and C═C bonds, the present inventors investigated whether these enzymes could be engineered to mimic this chemistry for isoelectronic carbene transfer reactions via a high-valent iron-carbenoid species (FIG. 1). This example demonstrates that variants of the cytochrome P450 from Bacillus megaterium (CYP102A1, or P450_(BM3)) are efficient catalysts for the asymmetric cyclopropanation of styrenes and other olefins.

Because iron porphyrins have been reported to catalyze carbene-based cyclopropanations (J. R. Wolf et al., J. Am. Chem. Soc. 117, 9194 (1995); B. Morandi et al., Science 335, 1471 (2012)), whether some common heme proteins display measurable levels of cyclopropanation activity in aqueous media (phosphate buffer, 5% methanol co-solvent) was first probed. The reaction between styrene and ethyl diazoacetate (EDA) was chosen (FIG. 2), a well-recognized model system for validating new cyclopropanation catalysts. The experiments showed that optimal formation of the desired cyclopropanation products occurred in the presence of a reducing agent (e.g., sodium dithionite, Na₂S₂O₄) under anaerobic conditions (Tables 7-10). Horseradish peroxidase (HRP), cytochrome c (cyt c), myoglobin (Mb) and P450_(BM3) all displayed multiple turnovers towards the cyclopropane products, with HRP, cyt c and Mb showing negligible enantioinduction and formed the trans cyclopropane with over 90% diastereoselectivity, which is comparable to the diastereoselectivity induced by free hemin (Table 7). P450_(BM3), despite forming the cyclopropane products in low yield, catalyzed the reaction with different diasteroselectivity (cis:trans 37:63) and slight enantioinduction (Table 28), showing that carbene transfer and selectivity are dictated by the heme cofactor bound in the enzyme active site.

TABLE 28 Stereoselective P450_(BM3) cyclopropanation catalysts. Reactions were run in aqueous phosphate buffer (pH 8.0) and 5% MeOH cosolvent at room temperature under argon with 30 mM styrene, 10 mM EDA, 0.2 mole % catalyst (with respect to EDA), and 10 mM Na₂S₂O_(4.) Catalyst % yield TTN cis:trans % ee_(cis)* % ee_(trans)† Hemin 15 73  6:94 −1 0 P450_(BM3) 1 5 37:63 −27 −2 P450_(BM3)-T268A 65 323  1:99 −15 −96 9-10A-TS-F87V 1 7 35:65 −41 −8 H2-5-F10 59 294 16:84 −41 −63 H2A10 33 167 60:40 −95 −78 H2-4-D4 41 206 53:47 −79 −33 BM3-CIS 40 199 71:29 −94 −91 BM3-CIS-I263A 38 190 19:81 −62 −91 BM3-CIS-A328G 37 186 83:17 52 −45 BM3-CIS-T438S 59 293 92:8  −97 −66 BM3-CIS-C400S 29 150 93:7  −99 −51 Yields, diastereomeric ratios, and enantiomeric excess were determined by GC analysis. Yields based on EDA. TTN = total turnover number. *(R,S)-(S,R). † (R,R)-(S,S). See, below for protein sequences indicating mutations from wild type P450_(BM3). Variant 9-10A-TS-F87V-T268A (herein called BM3-CIS).

The activity levels exhibited by the native proteins tested are not suitable for practical synthetic applications. Whether the activity and selectivity of heme-catalyzed cyclopropanation could be enhanced by engineering the protein sequence was therefore explored. P45_(BM3) is a well-studied, soluble, self-sufficient (heme and diflavin reductase domains are fused in a single polypeptide, ˜120 KDa), long-chain fatty acid monooxygenase. More than a decade of protein engineering attests to the functional plasticity of this biocatalyst (C. J. C. Whitehouse et al., Chemical Society Reviews 41, 1218 (2012)). Thousands of variants that exhibit monooxygenase activity on a wide range of substrates have been accumulated from the use of engineered cytochrome P450_(BM3) for synthetic applications (J. C. Lewis et al., Chimia 63, 309 (2009)). Some of these variants were tested for altered cyclopropanation diastero- and enantioselectivity by analysis of product distributions using gas chromatography (GC) with a chiral stationary phase. A panel of 92 P450_(BM3) variants, chosen for diversity of activity and protein sequence, was screened in E. coli lysate for the reaction of styrene and EDA under aerobic conditions in the presence of Na₂S₂O₄ (Tables 12 and 13). The ten most promising hits were selected for purification and characterization under standardized anaerobic reaction conditions (Tables 14 and 28).

Five of the ten selected P450s showed improvements in activity compared to wild type (total turnover numbers (TTN)>100), a comprehensive range of diastereoselectivities with cis:trans ratios varying from 9:91 to 60:40, and up to 95% enantioselectivities (Table 14). For example, variant H2-5-F10, which contains 16 amino acid substitutions, catalyzes 294 total turnovers, equivalent to ˜58% yield under these conditions (0.2% enzyme loading with respect to EDA). This represents a 50-fold improvement over wild type P450_(BM3). Furthermore, mutations affect both the diastereo- and enantioselectivity of cyclopropanation: H2-5-F10 favors the trans cyclopropanation product (cis:trans 16:84) with 63% ee_(trans), whereas H2A10, with a TTN of 167, shows reversed diastereoselectivity (cis:trans 60:40) with high enantioselectivity (95% ee_(cis)).

H2A10 was used to verify the role of the enzyme in catalysis and identify better reaction conditions (Table 15, FIGS. 3 and 4). Heat inactivation produced diastereo- and enantioselectivities similar to those obtained with free hemin, consistent with protein denaturation and release of the cofactor. Complete inhibition was achieved by pre-incubating the reaction mixture with carbon monoxide, which irreversibly binds the reduced P450 heme, confirming that catalysis occurs at the active site. Air inhibited the cyclopropanation reaction by about 50%, showing that dioxygen and EDA compete for reduced Fe^(II). Cyclopropanation was also achieved with NADPH as the reductant, confirming that the activity can also be driven by the endogenous electron transport machinery of the diflavin-containing reductase domain. The presence of a reducing agent in sub-stoichiometric amounts proved essential for cyclopropanation (Table 16), implying that the active species is Fe^(II) rather than the resting state Fe^(III).

Highly active P450_(BM3) variants H2A10, H2-5-F10 and H2-4-D4 have three to five active site alanine substitutions with respect to 9-10A-TS-F87V (12 mutations from P450_(BM3)), which itself shows negligible cyclopropanation activity. These variants exhibit a range of TTN, diastereoselectivity, and enantioselectivity (Table 28). To better understand how protein sequence controls P450-mediated cyclopropanation, 12 variants were constructed to assess the contributions of individual alanines to catalysis and stability (Table 17). T268A is key for achieving high cyclopropanation activity, and this mutation alone converts inactive 9-10A-TS-F87V into an active cyclopropanation catalyst. Variant 9-10A-TS-F87V-T268A (herein called BM3-CIS) is a competent cyclopropanation catalyst (199 TTN), displays strong preference for the cis product (cis:trans 71:29), forms both diastereomers with over 90% ee, and is as stable as wild-type P450_(BM3). Other active site alanine mutations tune the product distribution. Notably, the addition of I263A to BM3-CIS reverses diastereoselectivity (cis:trans 19:81). The effects of similar mutations introduced in the poorly active wild type P450_(BM3) were also investigated (Table 18). Impressively, P450_(BM3)-T268A, with a single mutation, is an active cyclopropanation catalyst (323 TTN, Table 28) with exquisite trans-selectivity (cis:trans 1:99) and high enantioselectivity for the major diastereomer (−96% ee_(trans), Table 28). Whereas BM3-CIS is a cis-selective cyclopropanation catalyst, identical active site mutations in wild type P450_(BM3) result in a trans-selective enzyme (Table 18), demonstrating that mutations outside of the active site also influence the stereochemical outcome.

Because the design of cis-selective small-molecule catalysts for diazocarbonyl-mediated cyclopropanations has proven more challenging than their trans counterparts (A. Caballero et al., European Journal of Inorganic Chemistry, 1137 (2009)), whether active site engineering of P450_(BM3) could provide robust cis-selective water-compatible catalysts to complement existing organometallic systems was investigated (I. Nicolas et al., Coordination Chemistry Reviews 252, 727 (2008)). Five active site residues (L181, I263, A328, L437, T438) were chosen for individual site-saturation mutagenesis. The A328G, T438A, T438S and T438P variants exhibited enhanced cis-selectivity (Table 19). Notably A328G also reversed the enantioselectivity for the cis-diastereomer (Table 28). BM3-CIS-T438S displayed the highest diastereo- and enantioselectivities (cis:trans 92:8 and −97% ee_(cis)) and maintained TTN comparable to BM3-CIS (Table 28).

Variant 9-10A-TS-F87V-T268A (BM3-CIS) exhibits Michaelis-Menten kinetics (FIG. 5 and Table 20) with relatively high K_(M) values for the olefin (˜1.5 mM) and the diazoester (˜5 mM), reflecting the lack of evolutionary pressure for this enzyme to bind these substrates. BM3-CIS exhibits a notable C_(cat) for cyclopropanation of 100 min⁻¹, comparable to the k_(cat) of many native P450s for hydroxylation, but about fifty times less than P450_(BM3)-catalyzed fatty acid hydroxylation (Table 21). Free hemin does not exhibit saturation kinetics and displays slower initial rates than BM3-CIS (only 30 min⁻¹ at 10 mM styrene and 15 mM EDA), indicating that the protein scaffold enhances k_(cat) compared to the free cofactor in solution. When used at 0.2 mol % equivalent, BM3-CIS-catalyzed cyclopropanations reached completion after 30 minutes. Adding more EDA enhanced turnovers for cyclopropanes and preserved variant 9-10A-TS-F87V-T268A (BM3-CIS) stereoselectivity (Table 22), confirming catalyst integrity and implying that the reaction stops because of EDA depletion rather than protein inactivation.

This example shows that different variants of this enzyme will accept a wide range of substrates for cyclopropanation. To begin to assess the substrate scope of P450_(BM3)-catalyzed cyclopropanation, the activities of six variants were investigated against a panel of olefins and diazo compounds (Tables 29-34).

TABLE 29 Scope of P450 catalyzed cyclopropanation of styrenyl substrates.

Reagents P450 catalyst TTN Z:E % ee_(Z) % ee_(E) R₁ = H, X = Me, R₂ = Et BM3-CIS 228 78:22 −81 N/A R₁ = H, X = OMe, R₂ = Et H2-5-F10 364 11:89   38 N/A R₁ = H, X = CF₃, R₂ = Et 7-11D 120 76:24   31 59 R₁ = Me, X = H, R₂ = Et 7-11D 157 41:49   42 N/A R₁ = H, X = H, R₂ = t-Bu H2A10 120  3:97 N/A N/A Ar = p-X—C₆H₄. Reaction conditions: 20 μM catalyst, 30 mM olefin, 10 mM diazoester, 10 mM Na₂S₂O₄, under argon in aqueous potassium phosphate buffer (pH 8.0) and 5% MeOH cosolvent for 2 hours at 298K. Enzyme loading is 0.2 mol % with respect to diazoester. N/A = not available when enantiomers did not separate to baseline resolution. Variant 9-10A-TS-F87V-T268A (herein called BM3-CIS).

TABLE 30 Substrate scope of P450 cyclopropanation catalysts: p-methylstyrene + EDA.

% % ee % ee P450 yield TTN cis:trans cis trans* 7-11D 21 104 54:46 0.3 N/A H2-5-F10 44 222 11:89 14.9 N/A C2G9 R1 18 92 10:90 8.9 N/A H2A10 10 50 43:57 −84.3 N/A 9-10A-TS-F87V- 46 228 78:22 −81.4 N/A T268A (BM3-CIS) BM3-CIS-C400S 32 157 93:7  −87.1 N/A Hemin 7 37  6:94 −1.6 N/A GC (cyclosil-B column 30 m × 0.32 mm, 0.25 μm film): oven temperature = 100° C. for 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution times: cis-cyclopropanes (21.03 and 21.18 min), trans-cyclopropanes (22.71 min). *trans-enantiomers did not resolve.

TABLE 31 Substrate scope of P450 cyclopropanation catalysts: p-vinylanisole + EDA

% % ee % ee P450 yield TTN cis:trans cis trans* 7-11D 59 297 70:30 −27 N/A H2-5-F10 73 364 11:89 38 N/A C2G9 R1 39 196 10:90 −1 N/A H2A10 16 80 40:60 −75 N/A 9-10A-TS-F87V- 43 214 48:52 −44 N/A T268A (BM3-CIS) BM3-CIS-C400S 47 235 81:19 −61 N/A hemin 19 96  7:93 0 N/A GC oven temperature = 110° C. for 8 min, 2° C./min to 180° C. then 180° C. for 30 min, 175 kPa. Cyclosil-B column (30 m × 0.25 mm, 0.25 μm film). Elution times: cis-cyclopropanes (38.74 and 39.52 min), trans-cyclopropanes (43.07 min). *Baseline resolution could not be achieved for the trans-enantiomers.

TABLE 32 Substrate scope of P450 cyclopropanation catalysts: p-(trifluoromethyl)styrene.

% % ee % ee P450 yield* TTN* cis:trans cis trans 7-11D 24 120 76:24 31 59 H2-5-F10 40 198 26:74 72 −65 C2G9 R1 18 89 10:90 4 0 H2A10 9 47 26:74 −24 22 9-10A-TS-F87V- 42 211 39:61 54 −93 T268A (BM3-CIS) BM3-CIS-C400S 24 121 76:24 55 −75 hemin 2 9 11:89 1 1 *Assumed the same detector response factor as for ethyl 2-(4-methylphenyl)cyclopropane-1-carboxylate. GC (cyclosil-B column (30 m × 0.25 mm, 0.25 μm film): oven temperature = 110° C. for 8 min, 2° C./min to 180° C. then 180° C. for 30 min, 175 kPa. Elution times: cis-cyclopropanes (27.26 and 28.11 min), trans-cyclopropanes (30.78 and 30.99 min).

TABLE 33 Substrate scope of P450 cyclopropanation catalysts: α-methyl styrene.

% % ee % ee P450 yield TTN Z:E (Z) (E) * 7-11D 31 157 41:49 42 N/A H2-5-F10 66 329 21:79 −14 N/A C2G9 R1 77 387 16:84 −4 N/A H2A10 34 168 19:81 −31 N/A 9-10A-TS-F87V- 26 127 16:84 −6 N/A T268A (BM3-CIS) WT F87V T268A 62 312  7:93 3 N/A BM3-CIS-C400S 17  86 30:70 34 N/A hemin 15  77 24:76 0 N/A GC oven temperature = 100° C. for 5 min, 1° C./min up to 135° C., 135° C. for 10 min, 10° C./min up to 200° C., 200° C. for 5 min. Cyclosil-B column (30 m × 0.32 mm, 0.25 μm film). Elution times: Z-cyclopropanes (34.96 and 35.33 min), E-cyclopropanes (39.34 and 39.61 min). * trans-enantiomers did not separate to baseline resolution.

TABLE 34 Substrate scope of P450 cyclopropanation catalysts: t-butyl diazoacetate.

P450 % yield* TTN* cis:trans WT F87V T268A 1.4 7 4:96 7-11D 11 54 13:87  H2-5-F10 18 90 3:97 H2A10 24 120 3:97 9-10A-TS-F87V-T268A 0.3 2 3:97 (BM3-CIS) BM3-CIS-C400S 15 76 8:92 hemin 20 100 4:96 *Assumed the same detector response factor as for ethyl 2-(4-methylphenyl)cyclopropane-1-carboxylate. GC (cyclosil-B column (30 m × 0.32 mm, 0.25 μm film): oven temperature = 100° C. for 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution times: cis-cyclopropanes (21.66 min), trans-cyclopropanes (23.31 min). Cis- and trans-enatiomers did not resolve.

P450 cyclopropanation is robust to both electron-donating (p-vinylanisole, p-vinyltoluene) and electron-withdrawing (p-trifluoromethylstyrene) substitutions on styrene, and variant 7-11D showed consistent cis-selectivity for these substrates. The P450s were also active on 1,1-disubstituted olefins (e.g., α-methyl styrene), with chimeric P450 C2G9R1 forming cyclopropanes in 77% yield (with respect to EDA). The P450s were only moderately active with t-butyl diazoacetate as substrate (<30% yield), forming the trans product with >87% selectivity and offering no advantage over free hemin (Table 34). For reactions involving EDA and aryl-substituted olefins, however, the P450s consistently outperformed the free cofactor in both activity and stereoselectivity. An appropriate catalyst for a given substrate can be found by testing the substrate against engineered or native P450s, as demonstrated above. Directed evolution methods well known to those of skill in the art can be used to enhance catalyst activity.

P450-derived cyclopropanases containing both heme and reductase domains remain competent monooxygenases, preferentially producing styrene oxide in the presence of NADPH under air. The possibility of generating a ‘specialist’ cyclopropanase that has no promiscuous monooxygenase activity was investigated. It is known that proximal cysteinate ligation (by C400 in P450_(BM3)) is important for dioxygen activation and stabilization of compound I during monooxygenation (J. H. Dawson, Science 240, 433 (1988)). As evidenced by the ability of free hemin to catalyze cyclopropanation, this ligand is likely not required for carbene transfer reactions, although it is required for the enzyme to catalyze monooyxgenation reactions. Site-saturation mutagenesis of C400 in wild-type P450_(BM3) revealed that only the isosteric C400S mutation led to folded heme-bound protein. Since this mutation has been reported to abolish monooxygenation activity in mammalian P450s (K. P. Vatsis et al., Journal of Inorganic Biochemistry 91, 542 (2002)), BM3-CIS-C400S was created, which remains an active cyclopropanase that is able to initiate the catalytic cycle by utilizing electrons from either dithionite (150 TTN) or NADPH (304 TTN). Unexpectedly, BM3-CIS-C400S displays considerably improved diastereo-(cis:trans/dithionite 93:7; NADPH 72:28) and enantioselectivity (dithionite −99% ee_(cis); NADPH 94% ee_(cis)) compared to its cysteine homologue (Tables 35-36 and FIG. 6) and is a much more active NADPH-driven cyclopropanase (FIG. 7).

TABLE 35 Effect of C400S mutation on BM3-CIS-mediated cyclopropanation driven by Na₂S₂O₄.

O₂ inhi- Yield bition % ee % ee P450 Conditions (%)^(a) TTN (%) cis:trans^(b) cis^(c) trans^(d) BM3- Anaerobic 29 143 — 77:23 −94 −91 CIS_(heme) BM3- Aerobic 11 55 −62 65:35 −87 −86 CIS_(heme) BM3-CIS- Anaerobic 30 151 — 93:7  −99 −51 C400S_(heme) BM3-CIS- Aerobic 0.6 3 −98 45:55 −79 −31 C400S_(heme) ^(a)based on EDA. ^(b)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(c)(R,S) − (S,R). ^(d)(R,R) − (S,S). Variant 9-10A-TS-F87V-T268A (herein called BM3-CIS).

TABLE 36 In vitro activities for purified P411_(BM3)-CIS vs P450_(BM3)-CIS driven by NADPH.

TTN TTN styrene O₂ cyclopropanes oxide TTN_(cyc)/ inhibition % ee % ee Cat. Conditions (TTN_(cyc)) (TTN_(epo)) TTN_(epo) (%) cis:trans^(a) cis^(b) trans^(c) P450_(BM3)- Anaerobic 60 ± 18 12 ± 10 5   — 60:40 −89 −53 CIS P450_(BM3)- Aerobic 82 ± 13 406 ± 21  0.20 +36 56:44 −88 −58 CIS P411_(BM3)- Anaerobic 304 ± 15  0 — — 72:28 −92 −19 CIS P411_(BM3)- Aerobic 43 ± 5  14 ± 2  3.1  −86 49:51 −74 −14 CIS ^(a)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(b)(R,S) − (S,R). ^(c)(R,R) − (S,S). Small-scale reactions (400 μL total volume) were conducted as described below with the following modifications: glucose dehydrogenase (GDH, 4 μL, 225 U mL⁻¹) was added to the reaction vial together with the P450 solution. Glucose (40 μL, 250 mM) and NADPH (40 μL, 5 mM) were degassed together with the buffer solution. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. The small amounts of epoxide formed by P450_(BM3)-CIS under anaerobic conditions are due to dioxygen contamination in the small-scale reactions in the experiments. Variant 9-10A-TS-F87V-T268A (herein called BM3-CIS).

Under aerobic conditions in the presence of NADPH, BM3-CIS-C400S forms negligible amounts of styrene oxide and is still able to form cyclopropanes (43 TTN), in marked contrast to BM3-CIS, which forms styrene oxide as the major product (FIG. 7). Cyclopropanation activity in BM3-CIS-C400S remains, however, severely inhibited by dioxygen (FIG. 7). It is noteworthy that Vatsis et al. have shown that the mammalian P450 CYP2B4-C436S which also has its proximal cysteine mutated to a serine is able to use electrons from NADH to reduce dioxygen to hydrogen peroxide. In particular, Vatsis et al. have shown that serine-heme P450s can function as a two-electron oxidase, accepting electrons from NADPH and reducing dioxygen to hydrogen peroxide. This peroxide generation activity can therefore be in competition with cyclopropanation under aerobic conditions.

BM3-CIS-C400S exhibits Michaelis-Menten kinetics (FIG. 8 and Table 37) with similar K_(M) values for the olefin (˜ 1.5 mM) and the diazocarbene (˜5 mM) as variant 9-10A-TS-F87V-T268A (BM3-CIS). Replacement of axial thiolate ligation with serine decreases the k_(cat) to about 20 min⁻¹.

TABLE 37 Michaelis-Menten parameters for P450 cyclopropanases. k_(cat)/ k_(cat)/ k_(cat)/(K_(M-EDA×) k_(cat) K_(M-EDA) K_(M-styrene) K_(M-EDA) K_(M-styrene) K_(M-styrene)) Catalyst (min⁻¹) (mM) (mM) (s⁻¹ M⁻¹) (s⁻¹ M⁻¹) (s⁻¹ M⁻¹ M⁻¹) BM3- 100.7 ± 5.2 ± 3.5 1.4 ± 0.5 321 1,111 2.1 × 10⁵ CIS_(heme) 24.1 BM3-CIS- 20.4 5.7 4.7 59.6 72.3 1.3 × 10⁴ C400S

BM3-CIS-C400S demonstrated consistent cis-selectivity for the substrates shown in Tables 30-34. In vitro cyclopropanation reactions catalyzed by BM3-CIS-C400S can be driven by sub-stoichiometric amounts of either NADH or NADPH.

To investigate whether the differences in stereoselectivity and catalytic rates caused by the C400S mutation were caused by changes in active site structure, the crystal structures of BM3-CIS and BM3-CIS-C400S were determined at 2.5 and 3.3 Å, respectively (FIG. 9 and Table 38).

TABLE 38 Data collection and refinement statistics for P450_(BM3) crystals. 9-10A-TS-F87V- T268A (BM3-CIS) BM3-CIS-C400S pdb accession # 4H23 4H24 Data collection* Space group I 1 2 1 P 2 21 21 wavelength 1.033 1.033 Cell dimensions a, b, c (Å) 187.79, 62.74, 210.28 63.16, 124.46, 127.69 αβγ (°) 90.00, 115.75, 90.00 90.00, 90.00, 90.00 Resolution (Å)  48.6-2.5 (2.5-2.6)**  44.9-3.3 (3.3-3.5)** R_(merge)  5.3 (39.5) 17.6 (51.4) I/σI 13.4 (3.0)  11.8 (5.7)  Completeness 98.7 (99.2) 99.9 (99.9) (%) Redundancy 2.6 (2.6) 5.3 (5.4) Refinement Resolution (Å) 48.6-2.5 44.9-3.3 No. reflections 72085 14884 R_(work)/R_(free) 0.19/0.25 0.18/0.26 No. atoms Protein 14401 6890 Ligand/ion 128 86 Water 196 24 B-factors Protein 33.9 25.4 Ligand/ion 25.4 19.9 Water 26.7 18.9 R.m.s. deviations Bond lengths (Å) 0.017 0.016 Bond angles (°) 1.70 1.65 Ramachandran 0.3% 0.7% outliers*** *All data sets were collected from single crystals. **Highest-resolution shell is shown in parentheses. ***Ramachandran outliers lie in regions of protein that are known to be flexible and show similar disorder among P450_(BM3) structures in the literature.

The structures are superimposable (RMSD=0.52 Å), with no significant changes in active site side chain or heme orientation. It is likely that changes in catalytic properties arise from electronic effects of altering the primary heme-ligand sphere. Both cis-selective BM3-CIS and BM3-CIS-C400S closely resemble the ligand-bound ‘closed’ form of P450_(BM3) (FIG. 9), while trans-selective P450_(BM3)-T268A resembles the open apo-form (J. P. Clark et al., Journal of Inorganic Biochemistry 100, 1075 (2006)). These large-scale conformational changes may contribute to the different diastereoselectivities observed among proteins that share nearly identical active site sequences.

Encouraged by the high turnovers achieved by BM3-CIS-C400S_(holo) in vitro when using NADPH as a reductant (FIG. 7), the catalytic cyclopropanation reaction was investigated using whole cells expressing cytochrome P450 variants. The stability of EDA was first assessed in the presence of E. coli cells (OD₆₀₀=30) lacking the P450_(BM3) gene and found no decomposition over the course of two hours. E. coli cells expressing BM3-CIS-C400S_(holo) are hereafter referred to as the ABC catalyst. Initial experiments to identify optimal conditions revealed that whole-cell reactions were also significantly inhibited by dioxygen (FIG. 10). The C400S mutation improves in vivo activity by a factor of 3-4 as can be seen in FIG. 10 by comparing the ABC catalyst with whole cells expressing the thiolate-ligated BM3-CIS_(holo). The ABC catalyst also affords higher diastereo- and enantioselectivity compared to BM3-CIS_(holo) (FIG. 10).

Addition of 2 mM glucose gave a consistent increase in cyclopropane yields (FIG. 11), presumably due to increased intracellular concentration of NADH, which is required to reduce the P450 catalyst to the active Fe(II) ferrous state. The ability of the whole cells to catalyze cyclopropanation to moderate yields even in the absence of exogenous glucose implies that some of the P450 is reduced with endogenous amounts of intracellular NAD(P)H. After normalizing for cell density, there are no significant differences in activity for cells grown in M9Y (M9, 1.5% yeast extract) or TB media. M9Y is the preferred growth medium due to its lower cost (FIG. 12). Expressing the holo enzyme gives a slightly higher yield of cyclopropanes compared to the heme domain alone (FIG. 12). Increasing ABC catalyst loading (cell density) increases cyclopropane yield up to approximately 80% at OD₆₀₀=50 (FIG. 13). When EDA is used at 10 mM, a 2:1 excess of styrene affords optimal yields (FIG. 14). Controls for whole cells containing the P450 gene but with no P450 induction and for whole cells devoid of the P450 gene are shown in FIG. 15.

The cell seems to stabilize the P450 catalyst, extending its activity for hours, such that the reaction only finishes when EDA is depleted, as shown in FIG. 16. Remarkably, under the conditions in FIG. 16, the in vivo TTN is over 8,000. At high substrate loading (100 mM EDA, 200 mM styrene) the in vivo TTN reaches over 30,000. This is approximately two orders of magnitude higher than the in vitro TTN values reported above for purified enzyme catalyst.

This example also shows that the ABC catalyst can be lyophilized and supplied as a solid that can be resuspended in the desired reaction conditions. It has been observed that the lyophilization process does not compromise the catalyst activity. Thus, this whole cell catalyst can be lyophilized and conveniently stored for long periods of time. It can be packaged, weighed out or otherwise distributed in solid form when needed for a reaction. Because it is genetically encoded in a whole cell, it can also be stored or distributed as a plasmid (to be transformed into live cells) or as the live cells (e.g., in a bacterial stab culture) and then grown to the desired density and volume, using methods well known to those of skill in the art.

In vivo catalysis offers some key advantages over the use of purified enzymes in vitro, such as the lower costs associated with catalyst preparation, the prolonged activity and the ability to incorporate the P450 cyclopropanation reaction into metabolic pathways.

This concept has been demonstrated for a single P450 enzyme, from Bacillus megaterium, and for chimeras of the B. megaterium enzyme with other, related P450s from B. subtilis. Those of skill in the art, however, will recognize that other P450s from other organisms can be engineered to carry out cyclopropanation and ABC whole-cell catalysts can be made using those enzymes. In particular, the equivalent of the C400S mutation will improve the performance of other P450 enzymes for cyclopropanation and other carbene transfer reactions. One of skill in the art knows how to identify the equivalent residue to C400 in other P450s, based on sequence alignments, an example of which is given below. Methods known in the art, such as site-directed mutagenesis or gene synthesis, can be used to alter this residue to serine in any P450. If the resulting enzyme folds properly, it will serve as a catalyst for cyclopropanation. This mutation in a purified protein or whole cell catalyst will improve the activity over the parent enzyme that does not include this mutation.

For example, BLAST alignment (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the amino acid sequence of P450_(BM3) (CYP102A1) to other P450s, such as the one from Pseudomonas putida (CYP101A1, P450_(CAM)) or the mammalian enzyme from Oryctolagus cuniculus (CYP2B4), enables identification of the proximal cysteine residue or of the equivalent T268 (marked in bold), as shown below:

CYP102A1 380 ENPSAIPQH--------AFKPFGNGQRACIGQQFALHEATLVL 414 E  +A P H        +   FG+G   C+GQ  A  E  + L CYP101A1 329 ERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTL 371 CYP102A1 265 GHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRL 324 G +T    LSF++ FL K+P   Q+  E   R     +P+               E LR CYP101A1 249 GLDTVVNFLSFSMEFLAKSPEHRQELIERPER-----IPA------------ACEELLR- 290 CYP102A1 374 FRPERF--ENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFD 422 F P  F   N +      F PF  G+R C+G+  A  E  L    +L++F CYP2B4 408 FNPGHFLDANGALKRNEGEMPFSLGKRVCLGEGIARTELFLFFTTILQNFS 458 CYP102A1 257 QIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVL-VDPVPSYKQVKQLKYVG 315 +++    AG ETTS  L +    ++K PHV ++  +E  +V+     P+     ++ Y CYP2B4 291 TVLSLFFAGTETTSTTLRYGFLLMLKYPHVTERVQKEIEQVIGSHRPPALDDRAKMPYTD 350

Therefore, the mutations C357S and T252A in CYP101A1 or C436S and T302A in CYP2B4 are expected to enhance the cyclopropanation activity in these enzymes. The mutation can be introduced into the target gene by using standard cloning techniques or by gene synthesis. The mutated gene can be expressed in the appropriate microbial host under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Cyclopropanation activity can be screened in vivo or in vitro by following product formation by GC or HPLC (see, Materials and Methods).

Because the ABC catalyst is genetically encoded and functions very well in whole cells, this catalyst can be incorporated into multi-enzyme pathways for biological synthesis in vivo, where multiple transformations of a substrate are carried out inside the cell. The EDA or other diazo reagent can be provided exogenously to the medium or generated in situ.

The ability to extend the C400S mutation to other P450 scaffolds allows access to a variety of diazo compounds as carbenoid precursors. These include, but are not limited to, diazo esters (acceptor type), diazo β-keto ester or β-cyano esters (acceptor-aceptor type), and alkyl,aryl, or alkenyl substituted diazo esters (donor-acceptor) (FIG. 17 a). These diazo compounds can be reacted inter- and intramolecularly with a variety of styrenes, aliphatic olefins, and allenes to provide biologically active compounds or organic building blocks for further reaction (FIG. 17 b). For instance, P450 catalyzed reaction of a diazo ester with aryl or allylic silanes and boronic acids would form cyclopropanes that can then be used in Suzuki or Hiyama cross coupling reactions. Furthermore, cyclopropanation of geranylacetone at the C9 olefin by a trans-selective P450 catalyst and EDA will provide a key precursor to anthroplalone and noranthroplone, marine natural products that exhibit microgram cytotoxicity against B-16 melanoma cells (FIG. 17 c) (W. A. Donaldson, Tetrahedron 57, 8589 (2001)). Lastly, treatment of 2,5-dimethylhexα-2,4-diene with a similar catalyst and EDA would provide the ethyl ester of chrysanthemic acid, an important intermediate in the production of pyrethroid insecticides (FIG. 17 d).

Materials and Methods

Unless otherwise noted, all chemicals and reagents for chemical reactions were obtained from commercial suppliers (Sigmα-Aldrich, Acros) and used without further purification. The following heme proteins were all purchased from Sigmα-Aldrich: myoglobin (from equine heart), peroxidase II (from horseradish), cytochrome c (from bovine heart), catalase (from Corynebacterium glutamicum) and chloroperoxidase (from Caldariomyces fumago). Silica gel chromatography purifications were carried out using AMD Silica Gel 60, 230-400 mesh. ¹H and ¹³C NMR spectra were recorded on either a Varian Mercury 300 spectrometer (300 MHz and 75 MHz, respectively), or a Varian Inova 500 MHz (500 MHz and 125 MHz, respectively), and are internally referenced to residual solvent peak. Data for ¹H NMR are reported in the conventional form: chemical shift (δ ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling constant (Hz), integration. Data for ¹³C are reported in terms of chemical shift (δ ppm) and multiplicity. High-resolution mass spectra were obtained with a JEOL JMS-600H High Resolution Mass Spectrometer at the California Institute of Technology Mass Spectral Facility. Reactions were monitored using thin layer chromatography (Merck 60 silica gel plates) using an UV-lamp for visualization. Optical rotation was measured using a JASCO P-2000 Polarimeter.

Gas chromatography (GC) analyses were carried out using a Shimadzu GC-17A gas chromatograph, a FID detector, and J&W scientific cyclosil-B columns (30 m×0.32 mm, 0.25 μm film and 30 m×0.25 mm, 0.25 μm film). High-performance liquid chromatography (HPLC) was carried out using an Agilent 1200 series, an UV detector, and an Agilent XDB-C18 column (4.6×150 mm, 5 μm). Cyclopropane product standards for the reaction of ethyl diazoacetate (EDA) with styrene (ethyl 2-phenylcyclopropane-1-carboxylate) and α-methylstyrene (ethyl 2-methyl-2-phenylcyclopropane-1-carboxylate) were prepared as reported (A. Penoni et al., European Journal of Inorganic Chemistry, 1452 (2003)). These standards and enzyme-prepared cyclopropanes demonstrated identical retention times in gas chromatograms when co-injected, confirming product identity. Absolute stereoconfiguration of cyclopropane enantiomers was determined by measuring optical rotation of purified cyclopropane products from preparative bioconversion reactions using enantioselective P450-BM3 variants and referenced to values taken from reference (N. Watanabe et al., Heterocycles 42, 537 (1996)). Authentic P450-catalyzed cyclopropane samples were also prepared as described herein and were characterized by NMR (¹H and ¹³C) and mass spectrometry.

Plasmids pCWori[BM3] and pET22 were used as cloning vectors. Site-directed mutagenesis was accomplished by standard overlap mutagenesis using primers bearing desired mutations (IDT, San Diego, Calif.). Electrocompetent Escherichia coli cells were prepared following the protocol of Sambrook et al., Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, New York, 1989), vol. 2. Restriction enzymes BamHI, EcoRI, XhoI, Phusion polymerase, and T4 ligase were purchased from New England Biolabs (NEB, Ipswich, Mass.). Alkaline phosphatase was obtained from Roche (Nutley, N.J.). The 1,000× trace metal mix used in expression cultures contained: 50 mM FeCl₃, 20 mM CaCl₂, 10 mM MnSO₄, 10 mM ZnSO₄, 2 mM CoSO₄, 2 mM CuCl₂, 2 mM NiCl₂, 2 mM Na₂MoO₄, and 2 mM H₃BO₃.

Enzyme Library Screening.

Libraries are stored at −78° C. as glycerol stocks (Luriα-Bertani medium (LB_(amp)), 150 μL, 25% v/v glycerol with 0.1 mg mL⁻¹ ampicillin) in 96-well plates. These stocks were used to inoculate 96-shallow-well plates containing 300 μL LB_(amp) medium using a 96-pin stamp. Single colonies from site-saturation libraries were picked with toothpicks and used to inoculate 300 μL of LB_(amp). The cells were incubated at 37° C., 250 rpm shaking, and 80% relative humidity overnight. After 16 h, 50 μL aliquots of these overnight cultures were transferred into 2 mL, deep-well plates containing terrific broth (TB_(amp)) (800 μL containing 0.1 mg mU⁻¹ ampicillin, 1 μL mL⁻¹ trace metal mix and 20 mg L⁻¹ aminolevulinic acid) using a Multimek 96-channel pipetting robot (Beckman Coulter, Fullerton, Calif.). The cultures were incubated at 37° C. for 3 h and 30 min, and 30 min after reducing the incubation temperature to 25° C. (250 rpm, 80% relative humidity), 50 μl, isopropyl β-D-1-thiogalactopyranoside (IPTG, 4.5 mM in TB_(amp)) was added, and the cultures were allowed to continue for another 24 h at 25° C. (250 rpm, 80% relative humidity). Cells were then pelleted (3,000×g, 15 min, 4° C.) and stored at −20° C. until further use, but at least for 2 h. For cell lysis, plates were allowed to thaw for 30 mM at room temperature and then cell pellets were resuspended in 275 μL phosphate buffer (0.1 M, pH=8.0, 0.65 mg mU¹ lysozyme, 10 mM magnesium chloride and 40 U mL⁻¹ DNAse I). The lysing cells were incubated at 37° C. for 1 h. Cell debris was separated by centrifugation at 5,000×g and 4° C. for 15 min. The resulting crude lysates were then transferred to 96-well microtiter plates for CO assays and to 2 mL deep-well plates for bioconversions.

CO Binding Assay.

Na₂S₂O₄ (160 μL, 0.1 M in phosphate buffer, 0.1 M, pH=8.0) was added to P450_(BM3) variants in cell lysate (40 μL). The absorbance at 450 and 490 nm was recorded using a Tecan M1000 UV/Vis plate reader, and the microtiter plates were placed in a vacuum chamber. The chamber was sealed, evacuated to approximately −15 in Hg, purged with CO gas, and incubated for 30 min. The plates were then removed and the absorbance at 450 and 490 nm was again recorded using a plate reader. The difference spectra could then be used to determine the P450 concentration in each well as previously described (C. R. Otey, in Methods in Molecular Biology: Directed Enzyme Evolution, F. H. Arnold, G. Georgiou, Eds. (Humana Press, Totowa, N.J., 2003), vol. 230).

P450 Expression and Purification.

For the enzymatic transformations, P450_(BM3) variants were used in purified form. Enzyme batches were prepared as follows. One liter TB_(amp) was inoculated with an overnight culture (100 mL, LB_(amp)) of recombinant E. coli DH5α cells harboring a pCWori plasmid encoding the P450 variant under the control of the tac promoter. After 3.5 h of incubation at 37° C. and 250 rpm shaking (OD₆₀₀ ca. 1.8), the incubation temperature was reduced to 25° C. (30 min), and the cultures were induced by adding IPTG to a final concentration of 0.5 mM. The cultures were allowed to continue for another 24 hours at this temperature. After harvesting the cells by centrifugation (4° C., 15 min, 3,000×g), the cell pellet was stored at −20° C. until further use but at least for 2 h. The cell pellet was resuspended in 25 mM Tris.HCl buffer (pH 7.5 at 25° C.) and cells were lysed by sonication (2×1 min, output control 5, 50% duty cycle; Sonicator, Heat Systems—Ultrasonic, Inc.). Cell debris was removed by centrifugation for 20 min at 4° C. and 27,000×g and the supernatant was subjected to anion exchange chromatography on a Q Sepharose column (HiTrap™ Q HP, GE Healthcare, Piscataway, N.J.) using an AKTAxpress purifier FPLC system (GE healthcare). The P450 was eluted from the Q column by running a gradient from 0 to 0.5 M NaCl over 10 column volumes (P450 elutes at 0.35 M NaCl). The P450 fractions were collected and concentrated using a 30 kDa molecular weight cut-off centrifugal filter and buffer-exchanged with 0.1 M phosphate buffer (pH=8.0). The purified protein was flash-frozen on dry ice and stored at −20° C. P450 concentration was determined in triplicate using the CO binding assay described above (10 μL P450 and 190 μL 0.1 M phosphate buffer, pH 8.0, per well).

Thermostability Measurements.

Duplicate measurements were taken for all values reported on Tables 17 and 18. Purified P450 solutions (4 μM, 200 μL) were heated in a thermocycler (Eppendorf) over a range of temperatures (38° C.-65° C.) for 10 min followed by rapid cooling to 4° C. for 1 min. The precipitate was removed by centrifugation. The concentration of folded P450 remaining in the supernatant was measured by CO-difference spectroscopy (as described above). The temperature at which half of the protein was denatured (T₅₀) was determined by fitting the data to the equation: f(T)=100/(1+exp(a*(T−T₅₀))).

Typical Procedure for Small-Scale Cyclopropanation and Carbene Insertion Bioconversions Under Anaerobic Conditions.

Small-scale reactions (400 μL) were conducted in 2 mL crimp vials (Agilent Technologies, San Diego, Calif.). P450 solution (80 μL, 100 μM) was added to the vial with a small stir bar before crimp sealing with a silicone septum. Phosphate buffer (260 μL, 0.1 M, pH=8.0) and 40 μL of a solution of the reductant (100 mM Na₂S₂O₄, or mM NADPH) were combined in a larger crimp-sealed vial and degassed by bubbling argon through the solution for at least 5 min (FIG. 3). In the meantime, the headspace of the 2 mL reaction vial with the P450 solution was made anaerobic by flushing argon over the protein solution (with no bubbling). When multiple reactions were conducted in parallel, up to 8 reaction vials were degassed in series via cannulae. The buffer/reductant solution (300 μL) was syringed into the reaction vial, while under argon. The gas lines were disconnected from the reaction vial before placing the vials on a plate stirrer. A 40× styrene solution in MeOH (10 μL, typically 1.2 M) was added to the reaction vial via a glass syringe, and left to stir for about 30 s. A 40×EDA solution in MeOH was then added (10 μL, typically 400 mM) and the reaction was left stirring for the appropriate time. The final concentrations of the reagents were typically: 30 mM styrene, 10 mM EDA, 10 mM Na₂S₂O₄, 20 μM P450.

The reaction was quenched by adding 30 μL HCl (3M) via syringe to the sealed reaction vial. The vials were opened and 20 μL internal standard (20 mM 2-phenylethanol in MeOH) was added followed by 1 mL ethyl acetate. This mixture was transferred to a 1.8 mL eppendorf tube which was vortexed and centrifuged (16,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by chiral phase GC.

A slightly modified work-up was implemented for kinetic experiments. The reactions were quenched after the set time by syringing 1 mL EtOAc to the closed vials and immediately vortexing the mixture. The vials were then opened and 20 μL internal standard was added. The mixture was transferred to a 1.8 mL eppendorf tube, vortexed and centrifuged (16,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by GC.

Typical Procedure for Preparative-Scale Cyclopropanation Bioconversions Under Anaerobic Conditions.

The P450 solution was added to a Schlenk flask with a stir bar. With the flask kept on ice, the head-space was evacuated and back-filled with argon (4×) with care not to foam the protein solution. Phosphate buffer and reductant were pre-mixed and degassed together in a separate round-bottom-flask by bubbling argon through the solution for 20 min. The buffer/reductant solution was transferred to the Schlenk flask via syringe. Styrene was added under argon and left to mix for 1 min. EDA was added dropwise under argon. The solution was left to stir under argon until reaction completion. The reaction was quenched under argon by adding hydrochloric acid (3 M) to adjust the pH to 4, before opening the Schlenk flask. The reaction mixture was stirred with sodium chloride and dichloromethane (CH₂Cl₂). The combined emulsion layers were then filtered through Celite to break the emulsion and the Celite pad was rinsed with 3×20 mL CH₂Cl₂. The resulting biphasic mixture was transferred to a separating funnel and the organic phase was removed. The remaining aqueous phase was re-extracted with 3×40 mL CH₂Cl₂. The combined organic extracts were dried with sodium sulfate, filtered, and concentrated. The resulting residue was purified by SiO₂ chromatography.

Supplementary Data

Preliminary Experiments with Heme Proteins

The following six heme proteins were initially screened for cyclopropanation activity: catalase, chloroperoxidase (CPO), horseradish peroxidase (HRP), cytochrome C (cyt c), myoglobin (Mb) and P450_(BM3). Small-scale (400 μL) reactions were conducted as described above and were analyzed by GC (cyclosil-B 30 m×0.25 mm×0.25 μm): oven temperature=130° C. Table 7 shows heme catalysts under anaerobic conditions with sodium dithionite (Na₂S₂O₄). Table 8 shows heme catalysts under anaerobic conditions without Na₂S₂O₄. Table 9 shows heme catalysts under aerobic conditions with Na₂S₂O₄. Table 10 shows heme catalysts under aerobic conditions without Na₂S₂O₄.

Screening P450_(BM3) Variants for Cyclopropanation Activity

Lysate screening under aerobic conditions. The 92 variants in the compilation plate (Table 12) represent a diverse selection of P450_(BM3) variants that have previously been engineered for monooxygenase activity on a variety of substrates, including but not limited to short alkane hydroxylation, demethylation of protected monosaccharides, and oxidation of lead drug compounds. These P450_(BM3) variants carry various mutations accumulated along sequential rounds of engineering efforts for activity towards the target substrates (Table 12) or were generated by recombination with homologous enzymes (Table 13). The compilation plate was expressed and lysed as described above. 150 μL lysate was transferred (Multimek 96-channel pipetting robot, Beckman Coulter, Fullerton, Calif.) to a 2 mL deep-well plate, with 50 μl, of 120 mM Na₂S₂O₄ in 0.1 M KPi (pH=8.0). 100 μL of a 30 mM styrene, 60 mM EDA mixed solution in 15% MeOH in 0.1 M KPi (pH=8.0) was added to the plate to initiate the reaction. The plate was sealed and was left shaking (300 rpm) for four hours. The plastic seal was removed and 30 μL HCl (3 M) was added to quench the reaction followed by 20 μL of an internal standard solution (20 mM α-methylstyrene in methanol). The reactions were extracted by adding 500 μL EtOAc and carefully vortexing the plate. The plate was centrifuged (1,700×g) to separate the biphasic mixture. The top organic layer was transferred (2×150 μL) to a separate deep-well plate. The extracts for each of the 92 reactions were dried through 92 separate anhydrous sodium sulfate plugs. The dried extracts were analyzed by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=60° C. 3 min, 7.5° C./min to 160° C., 20° C./min to 250° C., 250° C. 2 min, cis-cyclopropanes (20.3 min and 20.45 min), trans-cyclopropanes (21.8 min). The top 10 protein variants of importance with respect to this report are highlighted in Tables 12 and 13.

Determining the Cyclopropanation Activity of the Top 10 Hits (Highlighted on Tables 12 and 13) Under Anaerobic Conditions.

Small-scale reactions (400 μL total volume) were conducted as described above and were analyzed by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=100° C. 5 min, 1° C./min to 135° C., 135° C. 10 min, 10° C./min to 200° C., 200° C. 5 min, cis-cyclopropanes (39.40 min and 40.20 min), trans-cyclopropanes (44.69 min and 45.00 min). Table 14 shows stereoselective P450_(BM3)-based cyclopropanation catalysts.

Experimental Characterization of P450_(BM3) Cyclopropanation Catalysts

Controls to Confirm the Enzymatic Cyclopropanation Activity of Variant H2A10.

Small-scale reactions (400 μL total volume) were set up and worked up as described above. For the carbon monoxide (CO) inhibition experiment, the reaction vial and the buffer/reductant vial were purged with CO after having been purged with argon. For the boiled P450 experiment, a 100 μM solution of variant H2A10 was heated at 60° C. for 10 min. For the hemin experiment, hemin (80 μL) was added from a 1 mM solution in 50% DMSO-H2O, such that its final concentration in the reaction was 200 μM. Complete System=10 mM styrene, 20 mM EDA, 20 mM Na₂S₂O₄, 20 μM P450 (H2A10) under anaerobic conditions. The dried ethyl acetate extracts were analyzed by chiral phase GC, using 2-phenylethanol as an internal standard (injector temperature=300° C., oven temperature=100° C. for 5 min, 1° C./min ramp up to 135° C., 135° C. for 10 min, 10° C./min ramp up to 200° C., 200° C. for 5 min). Elution time: cis-cyclopropanes (39.40 min and 40.20 min), trans-cyclopropanes (44.69 min and 45.00 min). Table 15 shows controls for P450 based cyclopropanation using variant H2A1.

Optimizing Cyclopropanation Reaction Conditions for Variant H2A10.

Small-scale reactions (400 μL final volume) were set up and worked up as described above. The dried ethyl acetate extracts were analyzed by chiral phase GC, using 2-phenylethanol as an internal standard (injector temperature=300° C., oven temperature=100° C. for 5 min, 5° C./min ramp up to 200° C., 20° C./min ramp up to 250° C., 250° C. for 5 min). Elution time: cis-cyclopropanes (19.20 min and 19.33 min), trans-cyclopropanes (20.44 min). The reaction conditions that gave optimal yields of cyclopropanes (with respect to EDA) were: 30 mM styrene, 10 mM EDA and 20 μM P450 and were used in subsequent experiments.

Styrene Concentration.

FIG. 3 illustrates the effect of styrene concentration on cyclopropane yield.

P450 Concentration.

FIG. 4 illustrates the effect of P450 (H2A10) concentration on cyclopropane yield.

Dithionite Concentration.

Table 16 shows the effect of the concentration of Na₂S₂O₄ on cyclopropane yield.

Mutational Analysis of Active Site Alanine Substitutions in 9-10A TS F87V.

Table 17 shows a mutational analysis of alanine substitutions on 9-10A TS F87V.

Sequential Introduction of BM3-CIS Active Site Mutations in Wild-Type P450_(BM3).

Table 18 shows introducing BM3-CIS related active site mutations in wild-type P450_(BM3).

Active Site Saturation Mutagenesis of BM3-CIS_(heme)

Library Construction.

To simplify library construction and screening, only the BM3-CIS heme domain, which comprises residues 1-462 was used. This truncated enzymes lacks the P450 native reductase and exhibits similar activity and stereochemical control to the full length enzyme using Na₂S₂O₄ as a reductant, but not NADPH. P450 site-directed mutagenesis and site-saturation libraries were assembled from PCR fragments generated from oligonucleotides containing the desired codon mutation or a degenerate NNK (or for reverse primers, the reverse complement MNN; where N=A,T,G,C,K=G,T and M=A,C) codon, which codes for all 20 amino acids and the TAG stop codon. PCR fragments were assembled using either standard overlap extension PCR or through restriction cloning using the Type IIS restriction enzyme, B sal, depending on convenience.

Lysate screening under aerobic conditions. The compilation plate was expressed and lysed as described above. 150 μL lysate was transferred (Multimek 96-channel pipetting robot, Beckman Coulter, Fullerton, Calif.) to a 2 mL deep-well plate, with 50 μL of 120 mM Na₂S₂O₄ in 0.1 M KPi (pH=8.0). 100 μL of a 90 mM styrene, 30 mM EDA mixed solution in 15% MeOH in 0.1 M KPi (pH=8.0) was added to the plate to initiate the reaction. The plate was sealed and was left shaking (300 rpm) for four hours. The plastic seal was removed and 304HCl (3 M) was added to quench the reaction followed by 20 μL of an internal standard solution (20 mM 2-phenylethanol in methanol). Acetonitrile (400 μL) was added before carefully vortexing the plate. The plate was centrifuged (1,700×g), the supernatant was filtered (1 μm glass, 96 well filter plate, Pall) and transferred (150 μL) to a 96-well microtiter plate (Agilent). Reactions were analyzed by reverse-phase HPLC (210 nm): 50% acetonitrile-water, 1.0 mL min⁻¹, cis-cyclopropanes (7.6 min), trans-cyclopropanes (9.7 min). Hits were selected based on enhancement of cis-selectivity over parent BM3-CIS.

Determining the Cyclopropanation Activity of Hits from the Site-Saturation Libraries Under Anaerobic Conditions.

Small-scale reactions (400 μL total volume) were conducted as described above and were analyzed by GC (cyclosil-B 30 m×0.25 mm×0.25 μm): oven temperature=130° C., 175 kPa, cis-cyclopropanes (39.40 min and 40.20 min), trans-cyclopropanes (44.69 min and 45.00 min). Table 19 shows the cyclopropanation activity of selected BM3-CIS_(heme) active site variants.

Kinetic Characterization of BM3-CIS

Determination of initial rates. Both styrene and EDA concentrations were varied in the presence of the P450s expressed as the heme-domain (0.5 or 1.0 μM BM3-CIS_(heme)). Reactions were set up in phosphate buffer (pH=8.0) with Na₂S₂O₄ as the reductant at 298 K, and were worked-up as described above. Three time points were taken and used to determine the rate of product formation by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=100° C. 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution time: cis-cyclopropanes (19.20 min and 19.33 min), trans-cyclopropanes (20.44 min). Kinetic parameters were determined by fitting the data to the standard Michaelis-Menten model.

FIG. 5 illustrates the initial velocities plot for BM3-CIS_(heme). (A) EDA concentration was varied at a saturating concentration of styrene (30 mM). (B) Styrene concentration was varied at a fixed concentration of EDA (20 mM). Initial rates were computed as the slope of a zero-intercept linear fit of three different time points from independent reactions. Error bars correspond to 1-σ (68.3%) confidence intervals for the slope. Table 20 shows the Michaelis-Menten parameters for P450 cyclopropanation catalysts. Table 21 shows kinetic parameters for wild-type cytochrome P450s acting on their native substrates and for an engineered variant of P450_(BM3) (propane monooxygenase, PMO) acting on the non-native substrate propane. Table 22 shows the effect of EDA addition at t=30 min on BM3-CIS-catalyzed cyclopropanations.

Substrate Scope of P450 Cyclopropanation Catalysts

Small-scale reactions. Selected P450 catalysts were surveyed at a small-scale (400 μL total volume) for each combination of reagents (olefins and diazo esters). The small-scale anaerobic bioconversions were conducted as described above and were analyzed by GC. Table 30 shows the substrate scope of P450 cyclopropanation catalysts: p-methylstyrene+EDA. Table 31 shows the substrate scope of P450 cyclopropanation catalysts: p-vinylanisole+EDA. Table 32 shows the substrate scope of P450 cyclopropanation catalysts: p-(trifluoromethyl)styrene. Table 33 shows the substrate scope of P450 cyclopropanation catalysts: α-methyl styrene. Table 34 shows the substrate scope of P450 cyclopropanation catalysts: t-butyl diazoacetate.

Preparative-Scale Bioconversions.

These reactions were conducted anaerobically as described above.

Cyclopropanation of Styrene with EDA.

Prepared using 1.5 mmol styrene (3 equiv), 0.5 mmol EDA (1 equiv) and 1 μmol BM3-CIS_(heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 25 mg of the cis-cyclopropane (1) and 8 mg of a mixture of cyclopropanes with trans (2) in 5:1 excess over cis (Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007); C. J. Sanders et al., Tetrahedron: Asymmetry 12, 1055 (2001); M. Lenes Rosenberg et al., Organic Letters 11, 547 (2009)). Diagnostic data for the cis-cyclopropane 1: ¹H NMR (CDCl₃, 500 MHz): δ 7.28 (m, 4H), 7.21 (m, 1H), 3.89 (q, J=7.1 Hz, 2H), 2.60 (m, 1H), 2.10 (m, 1H), 1.73 (m, 1H), 1.35 (m, 1H), 0.99 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 170.99, 136.56, 129.31, 127.88, 126.63, 60.18, 25.47, 21.80, 14.02, 11.12; [α]²⁵ _(D)=−7.056° (c 0.83, CHCl₃). Diagnostic data for the trans-cyclopropane 2: ¹H NMR (CDCl₃, 500 MHz): δ 7.20 (m, 3H), 7.03 (m, 2H), 4.10 (q, J=7.1 Hz, 2H), 2.45 (m, 1H), 1.83 (m, 1H), 1.53 (m, 1H), 1.23 (m, 1H), 1.21 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 173.43, 140.13, 128.46, 126.55, 126.16, 60.72, 26.18, 24.20, 17.09, 14.27; [α]²⁵ _(D)=+199.2° (c 0.50, CHCl₃). MS (EI⁺) m/z: 190 (M⁺), 162 (PhCH(CH₂)CHCO₂ ⁺), 145 (PhCH(CH₂)CHCO⁺). The absolute configuration of compounds 1 and 2 was determined by comparison of the sign of their optical rotations with that reported (N. Watanabe et al., Heterocycles 42, 537 (1996)). The enantiomeric excess was determined to be 92% for the cis-cyclopropane and 88% for the trans-cyclopropane by GC.

Cyclopropanation of p-methylstyrene with EDA.

Prepared using 1.5 mmol styrene (3 equiv), 0.5 mmol EDA (1 equiv) and 1 μmol BM3-CIS_(heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 10 mg of the cis-cyclopropane (3) and 16 mg of a mixture of cyclopropanes with trans(4): cis/2:1 (Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007)). Diagnostic data for the cis-cyclopropane 3: ¹H NMR (CDCl₃, 500 MHz): δ 7.17 (d, J=8.0 Hz, 2H), 7.08 (d, J=8.0 Hz, 2H), 3.91 (q, J=7.1 Hz, 2H), 2.56 (m, 1H), 2.32 (s, 3H) 2.06 (m, 1H), 1.69 (m, 1H), 1.32 (m, 1H), 1.02 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 171.12, 136.12, 133.42, 129.14, 128.60, 60.17, 25.23, 21.68, 21.10, 14.08, 11.21. Diagnostic data for the trans-cyclopropane 4: ¹H NMR (CDCl₃, 500 MHz): δ 7.09 (d, J=8.0 Hz, 2H), 7.01 (d, J=8.0 Hz, 2H), 4.19 (q, J=7.1 Hz, 2H), 2.50 (m, 1H), 2.33 (s, 3H), 1.88 (m, 1H), 1.59 (m, 1H), 1.33 (m, 1H), 1.29 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 173.58, 137.04, 136.08, 129.12, 126.10, 60.66, 25.94, 24.06, 21.11, 16.96, 14.28. MS (Er) m/z: 204 (M⁺), 175 ([M-Et]⁺) 131 ([M-COOEt]⁺). The enantiomeric excess was determined to be 82% for the cis-cyclopropane by GC. Baseline resolution of the trans-enantiomers could not be achieved.

Cyclopropanation of p-methoxystyrene with EDA.

Prepared using 1.5 mmol styrene (3 equiv), 0.5 mmol EDA (1 equiv) and 1 μmol BM3-CIS_(heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 16 mg of the trans-cyclopropane (6) and 3 mg of a mixture of cyclopropanes with cis:trans 15:1 (Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007)). Diagnostic data for the trans-cyclopropane 6: 6.96 (m, 3H), 6.75 (m, 2H), 4.09 (q, J=7.1 Hz, 2H), 3.72 (s, 3H), 2.41 (m, 1H), 1.75 (m, 1H), 1.48 (m, 1H), 1.21 (t, J=7.1 Hz, 3H), 1.18 (m, 1H). MS (EI⁺) m/z: 220 (M⁺), 191 ([M-Et]⁺), 175 ([M-EtO]⁺), 147 ([M-COOEt]⁺). The enantiomeric excess was determined to be 38% for the cis-cyclopropane by GC. The trans-enantiomers did not resolve to baseline resolution.

Cyclopropanation of Styrene with t-Butyl Diazo Acetate.

Prepared using 0.75 mmol styrene (3 equiv), 0.24 mmol t-BuDA (1 equiv) and 0.5 μmol BM3-CIS_(heme) (0.002 equiv). The product was purified by SiO₂ chromatography (9:1 hexanes-diethyl ether) to give 9 mg of the trans-cyclopropane (8) (Y. Chen et al., Journal of Organic Chemistry 72, 5931 (2007); C. J. Sanders et al., Tetrahedron: Asymmetry 12, 1055 (2001)). Diagnostic data for the trans-cyclopropane 4: ¹H NMR (CDCl₃, 500 MHz): δ 7.20 (m, 2H), 7.12 (m, 1H), 7.02 (m, 2H), 2.36 (m, 1H), 1.76 (m, 1H), 1.45 (m, 1H), 1.40 (s, 9H), 1.16 (m, 1H); ¹³C NMR (CDCl₃, 125 MHz): δ 172.58, 140.52, 128.42, 126.32, 126.07, 80.57, 28.17, 25.75, 25.31, 17.08. MS (Er) m/z: 218 (M⁺), 145 ([M-OtBu]⁺).

Orthogonalization of Cyclopropanation and Monooxygenation Activities

FIG. 18 illustrates the UV-Vis absorption spectra of purified BM3-CIS_(heme) (3 μM, upper line) and BM3-CIS-C400S_(heme) (2 μM, lower line). Insert shows solutions of both proteins at approximately 1 mM.

Concentration of BM3-CIS-C400S was determined by the micro BCA™ assay (Thermo Scientific) using BM3-CIS as a standard. The serine-ligated cytochrome P450 displays a lower extinction coefficient (˜5× lower) and a Soret band that is blue-shifted by 14 nm. Magnetic circular dichroism and electronic absorption spectra of the substrate-free ferric, ferrous and CO-bound ferrous serine-ligated P450 has been reported elsewhere (R. Perera et al., Archives of Biochemistry and Biophysics 507, 119 (2011)).

Activity Under Anaerobic Vs Aerobic Conditions with Sodium Dithionite as the Reductant.

Table 35 shows the effect of C400S mutation on BM3-CIS-mediated cyclopropanation driven by Na₂S₂O₄. FIG. 6 shows the cyclopropanation activities of BM3-CIS and BM3-CIS-C400S driven by sodium dithionite under anaerobic and aerobic conditions. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value.

Activity Under Anaerobic Vs Aerobic Conditions with NADPH as the Reductant.

Small-scale reactions (400 tit total volume) were conducted as described above with the following modifications: glucose dehydrogenase (GDH, 4 μL, 225 U mL⁻¹) was added to the reaction vial together with the P450 solution. Glucose (40 μL, 250 mM) and NADPH (40 μL, 5 mM) were degassed together with the buffer solution. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Table 36 shows the effect of C400S mutation on BM3-CIS-mediated cyclopropanation driven by NADPH.

Determination of Initial Rates.

Both styrene and EDA concentrations were varied in the presence of the P450s expressed as the heme-domain (3.5 μM BM3-CIS-C400S_(heme)). Reactions were set-up in phosphate buffer (pH=8.0) with sodium dithionite as the reductant at 298 K, and were worked-up as described above. Three time points were taken and used to determine the rate of product formation by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=100° C. 5 min, 5° C./min to 200° C., 20° C./mM to 250° C., 250° C. for 5 min. Elution time: cis-cyclopropanes (19.20 min and 19.33 min), trans-cyclopropanes (20.44 min). Kinetic parameters were determined by fitting the data to the standard Michaelis-Menten model. FIG. 8 shows the initial velocities plot for BM3-CIS-C400S_(heme). (A) EDA concentration was varied at a saturating concentration of styrene (30 mM). (B) Styrene concentration was varied at a fixed concentration of EDA (20 mM). Table 37 shows the Michaelis-Menten parameters for P450 cyclopropanases.

X-Ray Crystallography Statistics

FIG. 9 shows active site and protein alignments of BM3-CIS with BM3-CIS-C400S and wild type P450_(BM3).

To investigate the nature of enhanced stereoselectivity in BM3-CIS-C400S, crystal structures of both proteins were determined to assess any structural changes that may have occurred due to the axial Cys→Ser mutation. The top panels shows alignments of BM3-CIS (green) and BM3-CIS-C400S (peach) with left, middle and right panels showing active site residues, the active site I-helix, and global protein fold, respectively. No significant structural changes were observed (RMSD 0.52 Å). Middle panels: Large variations are observed upon comparing BM3-CIS with the open (ligand-free) form of wild type BM3 (purple, taken from PDB#21J2, RMSD 1.2 Å). Pronounced rearrangements are observed in active site side chain residues (left) as well as rotations within the I-helix. Global movements are also observed in the N-terminal rich beta domain as well as F- and G-helices (right, marked by double headed arrows). These movements are consistent with well-known transitions that occur upon substrate binding and are important for native monooxygenation catalysis. Bottom panels: Alignment of BM3-CIS with a ligand-bound BM3 structure (cyan, taken from PDB#1JPZ, RMSD 0.52 Å) demonstrates that BM3-CIS and BM3-CIS-C400S mimic the closed protein conformation even in the absence of substrate.

Table 38 shows data collection and refinement statistics for P450_(BM3) crystals.

Summary of Mutations in P450_(BM3) Variants

Mutations in variant P450 cyclopropanation catalysts are reported with respect to wild-type P450_(BM3).

-   7-11D (P. Meinhold et al., Adv. Synth. Catal. 348, 763 (2006)):     R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G,     R255S, A290V, L353V, A82F, A328V -   9-10A TS (J. C. Lewis et al., Chembiochem: A European Journal of     Chemical Biology 11, 2502 (2010)): V78A, P142S, T175I, A184V, S226R,     H236Q, E252G, A290V, L353V, I366V, E442K -   H2A10: 9-10A TS+F87V, L75A, L181A, T268A -   H2-5-F10: 9-10A TS+F87V, L75A, I263A, T268A, L437A -   H2-4-D4: 9-10A TS+F87V, L75A, M177A, L181A, T268A, L437A -   BM3-CIS: 9-10A TS+F87V, T268A

Whole Cell Catalysts for In Vivo Cyclopropanation

Media and Cell Cultures.

E. coli cells were grown from glycerol stock overnight (37° C., 250 rpm) in 5 ml M9Y medium (1 L: 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 5.0 g NH₄C1, 0.24 g MgSO₄, 0.01 g CaCl₂, 1.5% yeast extract, 1 mL micronutrients, 0.1 mg mL⁻¹ ampicillin). The pre-culture was used to inoculate 45 mL of M9Y medium and this culture was incubated at 37° C., 250 rpm for 2 h and 30 min. At OD₆₀₀=1.2, the cultures were cooled to 25° C. and induced with IPTG (0.25 mM) and δ-aminolevulinic acid (0.25 mM). Cultures were harvested after 20 h and resuspended (OD₆₀₀=30) in nitrogen-free M9 medium (1 L: 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 0.24 g MgSO₄, 0.01 g CaCl₂, 1 mL micronutrients). The micronutrient solution contains 0.15 mM (NH₄)₆MO₇O₂₄, 20.0 mM H₃BO₃, 1.5 mM CoCl₂, 0.5 mM CuSO₄, 4.0 mM MnCl₂, and 0.5 mM ZnSO₄. Aliquots of the cell suspension were used for determination of the cell dry weight (cdw, 2 mL) and P450 expression level (4 mL).

Small-Scale Whole Cell Bioconversions.

E. coli cells (OD₆₀₀=30, 425 μL) were made anaerobic by bubbling argon through the cell suspension in a crimped 2 mL vial. A degassed solution of glucose (50 μL, 20 mM) was added to the cells before adding EDA (12.5 μL of a 400 mM solution in MeOH) and olefin (12.5 μL of a 1.2 M solution in MeOH). The reactions were stirred at room temperature for the appropriate and were worked up by adding 20 μL of the internal standard (20 mM 2-phenylethanol) and extracting with 1 mL ethyl acetate. The organic layer was dried over Na₂SO₄ before analyzing the product mixture by chiral phase gas chromatography.

FIG. 11 illustrates the effect of glucose addition on in vivo cyclopropanation of styrene. Reaction conditions: 20 mM styrene, 10 mM EDA, under argon in nitrogen-free medium and 5% MeOH cosolvent for 2 hours at 298 K (OD₆₀₀˜24). Total turnover=concentration of cyclopropanes (mM)/cell density (g cdw/L) in units of mmol/g cdw.

FIG. 12 illustrates the effect of media and comparison of holo vs heme forms of BM3-CIS-C400S on in vivo cyclopropanation of styrene. Reaction conditions: 20 mM styrene, 10 mM EDA, 2 mM glucose under argon in nitrogen-free medium and 5% MeOH cosolvent for 2 hours at 298 K (OD₆₀₀˜24).

FIG. 13 illustrates that increasing ABC catalyst loading (cell density) increases cyclopropanes yield up to approximately 80% at OD₆₀₀=50.

FIG. 14 illustrates the effect of styrene concentration on cyclopropane yield.

FIG. 15 illustrates controls for ABC catalyzed cyclopropanation.

FIG. 16 illustrates that ABC catalyst is active for 3 hours. At OD₆₀₀=25, the P450 concentration in 0.85 μM, such that TTN>8,000.

Example 3 In Vivo and In Vitro Olefin Cyclopropanation Catalyzed by Heme Enzymes

This example illustrates the use of heme containing enzymes to catalyze the conversion of olefins to various products containing one or more cyclopropane functional groups. In certain aspects, this example demonstrates novel variants of cytochrome P450_(BM3) (CYP102A1 or BM3) having an improved ability to catalyze the formal transfer of carbene equivalents from diazo esters to various olefins, making cyclopropane products with high stereoselectivity. Preferred variants include, but are not limited to, cytochrome P450_(BM3) mutants having C400S and T268A amino acid substitutions and engineered variants of other P450s having the equivalent substitutions. Axial serine heme ligation (C400S in BM3) in cytochrome P450s creates the homologous “cytochrome P411” family. Cytochrome P411s catalyze the cyclopropanation reaction in whole cells, sustaining over 10,000 total turnovers with high stereoselectivity, making the cyclopropane product with titers of over 20 g L⁻¹.

Introduction

Genetically programmed whole-cell biocatalysts are readily produced in simple growth media, do not require further purification or isolation and can be engineered with metabolic pathways for the elaboration of complex molecules (P. K. Ajikumar et al., Science 330, 70 (2010); P. J. Westfall et al., Proc. Natl. Acad. Sci. U.S.A. 109, E111 (2012); M. Kataoka et al., Appl Microbiol. Biotechnol. 62, 437 (2003)). The range of accessible transformations, however, is currently limited to the chemical repertoire of natural enzymes. Designing enzymes for non-natural reactions in vivo has been challenging due to the requirements for assembly of the functional catalyst, the compatibility of synthetic reagents in the cellular milieu, and cell permeability to allow substrate influx and product release. The catalysis of non-natural transformations inside cells will enable alternative metabolic routes to natural and artificial products, bio-based production of chemicals currently made using synthetic reactions, and will expand the chemical toolbox available for in vivo studies of cellular function (M. Boyce, C. R. Bertozzi, Nature Methods 8, 638 (2011)).

The preceding examples demonstrate that a few amino acid mutations in a bacterial cytochrome P450 monooxygenase can unlock significant cyclopropanation activity in vitro. Variants of P450_(BM3) from Bacillus megaterium (BM3) catalyze hundreds of turnovers of formal carbene transfers from diazoesters (e.g., ethyl diazoacetate, EDA) to olefins (e.g., styrene) in the presence of a reductant, forming cyclopropane products with high levels of diastereo- and enantioselectivity (P. S. Coelho et al., Science 339, 307 (2013)). Olefin cyclopropanation is widely used in the synthesis of fine chemicals (H. Lebel et al., Chem. Rev. 103, 977 (2003)), and state-of-the-art asymmetric organometallic catalysts are able to catalyze thousands to tens of thousands of turnovers (D. A. Evans et al., J. Am. Chem. Soc. 113, 726 (1991); H. M. L. Davies, C. Venkataramani, Org. Lett. 5, 1403 (2003); G. Maas, Chem. Soc. Rev. 33, 183 (2004)). BM3 variants may be suitable for in vivo catalysis because they are readily expressed in functional form and can catalyze non-natural carbene transfers without requiring artificial cofactors or posttranslational modifications. To initiate the catalytic cycle inside a cell, it is necessary to reduce the enzyme to the catalytically active ferrous-P450 with an endogenous reducing agent such as NAD(P)H. Based on consideration of heme ligation control of the P450 Fe^(III)/Fe^(II) reduction potential, a genetically-encoded “ABC” catalyst that catalyzes efficient and selective olefin cyclopropanation in intact cells was designed.

Results

Cytochrome P450-catalyzed cyclopropanations require substoichiometric (with respect to diazoester and olefin) reductant and proceed optimally under anaerobic conditions (P. S. Coelho et al., Science 339, 307 (2013)). This indicates that diazoester activation and carbene transfer involve a reduced P450-bound Fe^(II)-heme cofactor as opposed to the resting state Fe^(III)-heme (FIG. 19A). Active P450-derived cyclopropanation catalysts show marked preference for strong reducing agents such as sodium dithionite (E^(o)′=−660 mV, all potentials vs SHE) over native NAD(P)H (E^(o)′=−320 mV) (P. S. Coelho et al., Science 339, 307 (2013)). This indicates a limited substrate-induced low-spin (E^(o)′ Fe^(III/II)=−430 mV) to high-spin (E^(o)′ Fe^(III/II)=−290 mV) transition of the P450 heme-iron (T. W. B. Ost et al., Biochemistry 40, 13421 (2001)), which, while essential for monooxygenation, may not be achievable in this engineered system due to the poor affinity for the non-natural substrates (K_(M)˜5 mM) (P. S. Coelho et al., Science 339, 307 (2013)). It was hypothesized that raising the reduction potential of the resting state enzyme to facilitate NAD(P)H reduction would be important for enhancing Fe^(II) catalysis in vivo. Because the reduction potential of heme proteins can be tuned by axial ligand mutations (D. S. Wuttke, H. B. Gray, Curr. Opin. Struct. Biol. 3, 555 (1993); C. J. Reedy et al., Nucleic Acids Res. 36, D307 (2008)), the Fe^(III/II) potential was raised by substituting the axial cysteine thiolate in BM3 with the weakly donating serine alcohol (FIG. 19A). Furthermore, axial cysteinate ligation is essential for dioxygen activation and stabilization of the active ferryl-porphyrin cation radical oxidant (compound I, FIG. 1) during monooxygenation (J. H. Dawson, Science 240, 433 (1988)), and axial cysteine to serine substitutions abolish monooxygenation activity in mammalian P450s (K. P. Vatsis et al., J. Inorg. Biochem. 91, 542 (2002)). Because free hemin is also a (poor) cyclopropanation catalyst (P. S. Coelho et al., Science 339, 307 (2013)), an axial cysteine to serine mutation (C400S in BM3) maintains carbene transfer activity while eliminating monooxygenation activity.

The C400S mutation was introduced into a cis-selective cyclopropanation catalyst of the present invention, BM3-CIS (13 mutations from BM3; P. S. Coelho et al., Science 339, 307 (2013)), to contrast with the trans-selectivity observed with iron-porphyrins (J. R. Wolf et al., J. Am. Chem. Soc. 117, 9194 (1995)). BM3-CIS catalyzes hundreds of turnovers in the presence of dithionite in vitro and forms the ethyl 2-phenylcyclopropane-1-carboxylate product with 71% cis-selectivity and −94% enantiomeric excess (ee_(cis)) (P. S. Coelho et al., Science 339, 307 (2013)). Heme-serine ligation in BM3-CIS-C400S (hereafter called ABC-CIS) was confirmed by determining the crystal structures of the BM3-CIS and ABC-CIS heme domains at 2.5 and 3.3 Å, respectively (FIG. 9 and Table 38, PDB: 4H23 and 4H24); the structures are superimposable (RMSD=0.52 Å, FIG. 9). Despite the limited resolution of the ABC-CIS structure, simulated annealing omit maps generated in the absence of modeled heme and C400S show density consistent with heme coordination by a proximal amino acid side chain (FIGS. 19B and 20). UV-vis spectra for the green-brown ABC-CIS (FIGS. 21-23) provide further evidence for heme-serine ligation and are consistent with those reported for a Ser-ligated mammalian P450 (K. P. Vatsis et al., J. Inorg. Biochem. 91, 542 (2002); R. Perera et al., Arch. Biochem. Biophys. 507, 119 (2011)) (Table 39), marked by a ferrous carbon monoxide-bound complex at 411 nm.

TABLE 39 Comparison of λmax for ABC-CISheme and CYP2B4-C436S (Vatsis et al.). ABC-CIS_(heme) (nm) CYP2B4-C436S (nm) Ferric resting state 404 405 Ferrous 425 422 Ferrous-CO 411 413

Potentiometric redox titrations using the truncated heme domains of wild-type BM3, its C400S variant (referred to herein as “ABC”), BM3-CIS and ABC-CIS (FIGS. 19C and 24-27) showed that the C400S mutation raises the reduction potential of the resting state enzyme by +127 mV (E^(o)′ Fe^(III/II) _(ser)=−293 mV and −265 mV for ABC and ABC-CIS, respectively). This shift is similar in magnitude to that which occurs in BM3 upon substrate binding (T. W. B. Ost et al., Biochemistry 40, 13421 (2001)), and therefore allows ABC-CIS to be reduced by NAD(P)H even in the absence of substrate.

ABC-CIS (P411_(BM3-heme)-CIS) is an active dithionite-driven cyclopropanation catalyst in vitro, with Michaelis-Menten parameters (k_(cat)=82 min⁻¹, K_(M-styrene)=4.6 mM, K_(M-EDA)=5.7 mM, FIG. 8), comparable to those of BM3-CIS (P450_(BM3-heme)-CIS) Table 40).

TABLE 40 Michaelis-Menten parameters for P450 cyclopropanation catalysts. Error bars correspond to 99% confidence intervals for the fitted parameters. k_(cat)/ k_(cat)/ k_(cat)/ (K_(M-EDA×) K_(M-EDA) K_(M-styrene) K_(M-styrene)) k_(cat) K_(M-EDA) K_(M-styrene) (s⁻¹ (s⁻¹ (s⁻¹ M⁻¹ Catalyst (min⁻¹) (mM) (mM) M⁻¹) M⁻¹) M⁻¹) P450_(BM3-heme)- 100 ± 5.2 ± 1.4 ± 320 1,100 2.1 × 10⁵ CIS (5) 24 3.5 0.5 P411_(BM3-heme)-  82 ± 5.7 ± 4.6 ± 240 300 5.5 × 10⁴ CIS 15 2.9 2.4

ABC-CIS displays considerably improved diastereoselectivity (cis:trans 93:7) and enantioselectivity (−99% ee_(cis)) compared to its cysteine homologue (FIG. 28), an unexpected result given the similar active site geometries of the two catalysts (FIG. 9). For a variety of styrenyl substrates, ABC-CIS (P411_(BM3)-CIS) showed superior cis-selectivity relative to BM3-CIS (Table 41).

TABLE 41 Enhanced Z selectivity for ABC-CIS (P411_(BM3)-CIS) over BM3-CIS (P450_(BM3)-CIS).

heme Reagents domain % yield* TTN Z:E^(†) % ee Z^(‡) % ee E^(‡) R₁ = H, P450_(BM3)- 46 228 78:22 −81.4 N/A X = Me, CIS R₂ = Et P411_(BM3)- 32 157 93:7  −87.1 N/A CIS R₁ = H, P450_(BM3)- 43 214 48:52 −44 N/A X = OMe, CIS R₂ = Et P411_(BM3)- 47 235 81:19 −61 N/A CIS R₁ = H, P450_(BM3)- 42 211 39:61 54 −93 X = CF₃, CIS R₂ = Et P411_(BM3)- 24 121 76:24 55 −75 CIS R₁ = Me, P450_(BM3)- 26 127 16:84 −6 N/A X = H, CIS R₂ = Et P411_(BM3)- 17 86 30:70 34 N/A CIS R₁ = H, P450_(BM3)- 0.3 2  3:97 N/A N/A X = H, CIS R₂ = t-Bu P411_(BM3)- 15 76  8:92 N/A N/A CIS *Based on EDA. ^(†)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(‡)Enantiomeric excess is only reported when the enantiomers resolved to baseline resolution.

ABC-CIS shows increased activity compared to BM3-CIS when NADPH is used as the reductant under anaerobic conditions (FIG. 7 and Table 36). BM3-CIS only forms small amounts of cyclopropanes when NADPH is used and forms styrene oxide via monooxygenation as the major product under aerobic conditions. In contrast, ABC-CIS produces negligible amounts of styrene oxide and is still able to form cyclopropanes under aerobic conditions, albeit with lower yields (43 TTN) due to oxygen inhibition (FIG. 7). Dioxygen inhibition could be due to a two-electron oxidase activity as reported for CYP2B4-C436S (K. P. Vatsis et al., J. Inorg. Biochem. 91, 542 (2002)). NADH drives ABC-CIS-mediated cyclopropanation as efficiently as NADPH (Table 42), indicating that ABC-CIS is well suited for in vivo catalysis under anaerobic conditions where NADPH biosynthesis in E. coli does not take place.

TABLE 42 In vitro BM3-CIS-C400S (also called ABC-CIS or P411_(BM3)-CIS) cyclopropanation driven by Na₂S₂O₄, NADPH and NADH. [NADPH]/ [NADH]/ [Na₂S₂O₄]/ Yield cis: % ee % ee mM mM mM (%)* TTN trans^(†) cis^(‡) trans^(§) 0 0 0 1 3  54:46 — — 0 0 10 35 218 94:6 −99 −43 1 0 0 47 294 93:7 −98 −40 5 0 0 58 364  90:10 −97 −28 10 0 0 49 305 91:9 −98 −26 0 1 0 70 437 93:7 −98 −38 0 5 0 68 428 93:7 −98 −34 0 10 0 47 295 93:7 −98 −34 *Based on EDA. ^(†)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(‡)(2R, 1S) − (2S, 1R). ^(§)(2R, 1R) − (2S, 1S).

The efficiency of cyclopropanation using resting Escherichia coli [BL21(DE3)] cells grown in M9Y media [M9, 1.5% yeast extract] expressing variant 9-10A-TS-F87V-T268A (also called BM3-CIS or P450_(BM3)-CIS) and BM3-CIS-C400S (also called ABC-CIS or P411_(BM3)-CIS) was next investigated. Addition of glucose under anaerobic conditions significantly increased product yield (FIG. 29), presumably due to enhanced intracellular production of NADH. ABC-CIS catalyzes thousands of turnovers in vivo, is about four times more active than BM3-CIS in whole-cells, and provides the cyclopropane products with enhanced cis-enantioselectivity (Table 43, entries 1 and 2).

TABLE 43 Cyclopropanation activities for intact E. coli cells expressing engineered enzymes.

Cell [EDA] Density [P450] ee_(cis) ee_(trans) Entry Catalyst (mM) (g_(cdw)/L) (μM) Yield (%) TTN cis:trans (%)* (%)^(†) 1 BM3-CIS 8.5 7.7 3.7 42 950 22:78 −60 −22 (P450_(BM3)-CIS) 2 ABC-CIS 8.5 7.7 1.3 55 3,700 76:24 −96 −25 (P411_(BM3)-CIS) 3 ABC-CIS_(heme) 8.5 7.7 3.6 67 1,600 71:29 −95 −17 (P411_(BM3-heme)-CIS) 4 BM3 (P450_(BM3)) 8.5 13.4 4.8  0.9 15 25:75 −24 −21 5 ABC (P411_(BM3)) 8.5 8.1 1.5 50 2,900 13:87 −12  −8 6 ABC-CIS 8.5 8.4 1.8  0.6 30 20:80 −35 −20 (P411_(BM3)-CIS) + CO 7 ABC-CIS 170 8.4 1.8 72 67,800 90:10 −99 −43 (P411_(BM3)-CIS)^(‡) 8 ABC-CIS 200 20 3.2 78^(§) 48,800 88:12 −99 −35 (P411_(BM3)-CIS)^(‡) P411_(BM3) = P450_(BM3)-C400S. Reaction conditions were as follows: 2 eq styrene, 1 eq EDA, 0.2 eq glucose, E. coli whole-cells in aqueous nitrogen-free M9 minimal medium and 5% MeOH cosolvent under anaerobic conditions for twelve hours at 298K. Yields, diastereomeric ratios, and enantiomeric excess were determined by GC analysis. Yields based on EDA. TTN = total turnover number. *(2R,1S) − (2S,1R). ^(†)(2R,1R) − (2S,1S). ^(‡)Neat reagents were used without addition of MeOH; reactions were left for 24 h. ^(§)Isolated yield (1.63 g cyclopropanes). The data represent the averages of triplicate experiments. Standard errors are within 20% of the reported average.

The C400S mutation compromises protein expression such that ABC-CIS accounts for 2% of dry cell mass compared to 6% for BM3-CIS. The reduced expression is not due to decreased protein stability, as C400S contributes to increased thermostability in the purified ABC-CIS heme domain (ABC-CIS_(heme), FIG. 30). The holo enzyme, which contains both heme and diflavin reductase domains, is over two times more active on a molar basis than the heme domain alone (Table 43, entry 3), confirming that reduction to the ferrous state in vivo is important, but also showing that the reducing intracellular environment achieves heme reduction even in the absence of the reductase domain. The C400S mutation (ABC) improves the in vivo cyclopropanation activity of BM3 by over two orders of magnitude (Table 43, entries 4 and 5). Purified ABC is also an efficient NADH-driven cyclopropanation catalyst in vitro, whereas BM3 is barely active (Table 44).

TABLE 44 In vitro cyclopropanation activities of BM3 and ABC driven by NADH.

Yield % ee % ee Catalyst (%)* TTN cis:trans^(†) cis^(‡) trans^(§) BM3 (P450_(BM3)) 0.2   2 ± 0.5 17:83 −29 −21 ABC (P411_(BM3)) 36   364 ± 50  12:88  −5  −1 *Based on EDA. ^(†)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(‡)(2R,1S) − (2S,1R). ^(§)(2R,1R) − (2S,1S). Small-scale (500 μL) reactions were conducted as described herein with purified P450_(BM3) and P411_(BM3) catalysts. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value.

Both ABC-CIS and BM3-CIS whole cells are significantly inhibited by dioxygen (FIG. 31). Whole-cells containing the ABC-CIS gene but with no induction and whole cells devoid of the ABC-CIS gene are able to form small amounts of cyclopropanes, but do so with stereoselectivity similar to that of free hemin (FIG. 32) since free hemin and other heme proteins present in cells are also able to catalyze styrene cyclopropanation at low levels (P. S. Coelho et al., Science 339, 307 (2013)). Whole-cell ABC-CIS catalysts are as stereoselective as purified ABC-CIS in vitro at equivalent catalyst loading (vide infra), demonstrating that the overexpressed P411 enzyme favorably outcompetes background catalysis. In vivo cyclopropanation is strongly inhibited by carbon monoxide (Table 43, entry 6), which irreversibly binds ferrous heme, confirming that catalysis occurs in the enzyme active site. Yields could be increased to 80% by increasing the cell density up to OD₆₀₀=50 (FIG. 33). Reaction yield was only slightly improved by using excess styrene (FIG. 14). Lysate of cells expressing ABC-CIS and with NADH added retain only about 30% of the activity of the intact whole-cells and are not active in the absence of exogenous reductant (Table 45).

TABLE 45 Lysate activity compared to in vivo activity. [P411] Yield cis: % ee Catalyst Conditions (μM) (%)* TTN trans^(†) cis^(‡) ABC-CIS In vivo 1.0 44 5120 80:20 −96 (P411_(BM3)-CIS) ABC-CIS Lysate, no 1.0 0.6 55 67:33 −92 (P411_(BM3)-CIS) reductant ABC-CIS Lysate + 1.0 18 1780 80:20 −97 (P411_(BM3)-CIS) NADH ABC-CIS Lysate + 1.0 0.8 79 64:36 −86 (P411_(BM3)-CIS) dithionite *Based on EDA. ^(†)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(‡)(2R, 1S) − (2S, 1R).

Addition of dithionite inhibited ABC-CIS whole-cell reactions and was less efficient than NADH in driving the reaction in cell lysate (FIG. 15 and Table 45).

In order to provide a direct comparison of enzyme activity in vivo versus in vitro, both reactions were monitored at the same enzyme concentration over 8 hours (FIG. 34). On a molar basis, the in vivo catalyst showed almost 6 times higher TTN than the purified enzyme after 8 hours and retained the same stereoselectivity (75:25 cis:trans, −95% ee_(cis)). Both catalysts remained active over 6 hrs, indicating that the observed differences in yield and TTN are due to improved activity rather than enhanced catalyst stability in vivo. Gradual addition of EDA did not improve the reaction yield.

At high substrate loading (170 mM EDA, 400 mM styrene, added as neat reagents), more than 60,000 catalytic turnovers were observed in the in vivo reaction with ABC-CIS (Table 43, entry 7). ABC-CIS whole-cell reactions are readily scalable to make gram quantities of cyclopropanes with high stereoselectivity, product titer (27 g L⁻¹) and yield (78%, Table 46). No organic cosolvents are necessary, and the cyclopropane products can be readily obtained by extraction with organic solvent at the end of the reaction. Furthermore, the cells can be lyophilized with a cryoprotectant such as sucrose and stored as a powder for weeks at 4° C. without degradation of catalytic activity or diastereo- and enantioselectivity (Table 47).

TABLE 46 Cyclopropanation activities for intact E. coli cells expressing ABC-CIS (P411_(BM3)-CIS).

vol. sp. c_((substrate)) c_((carbene)) c_((cells)) c_((product)) c_((product)) Yield¹ time productivity Productivity [mM] [mM] [g/L] [mM] [g/L] [%] [h] [g/L/h] [g/g/h] 400 170 20 142 27 84% 12² 2.3² 0.19² Reaction conditions are those described in Table 43 except where noted. ¹Yield is calculated based on carbene conversion. ²Reaction was run overnight; actual reaction time may be shorter and hence productivity numbers are the worst possible scenario.

TABLE 47 Cyclopropanation activity of lyophilized ABC-CIS (P411_(BM3)-CIS) whole-cell catalysts. Total [Cell Turnover density] [P411] Yield (mmol cis: % ee % ee Catalyst (g_(cdw) L⁻¹) (μM) (%)* g_(cdw) ⁻¹) TTN trans^(†) cis^(‡) trans^(§) ABC-CIS 6.0 0.8 43 0.710 ± 5300 ± 67:33 −93 −25 (P411_(BM3)-CIS) 0.08 600 *Based on EDA. ^(†)Diastereomeric ratios and enantiomeric excess were determined by GC analysis. ^(‡)(2R, 1S) − (2S, 1R). ^(§)(2R, 1R) − (2S, 1S). Cells were lyophilized in 10% sucrose (m/V) and were stored at 4° C. for two weeks. An appropriate mass of the resulting powder was transferred to a 2 mL glass vial, which was crimp sealed and purged with argon. Degassed solutions of nitrogen-free M9 medium and glucose (20 mM) were added via syringe. Cells were resuspended to OD₆₀₀ = 25 and 2 mM final concentration of glucose. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value.

The lyophilized cells can be readily packaged and distributed. These features render the whole cell process attractive for facile benchtop synthesis.

ABC catalysts based on a Ser-ligated cytochrome-P411 are spectroscopically, electrochemically and catalytically distinct from cytochrome P450s. Whole-cell ABC catalysts are easy to use and deliver high conversion, optical purity and yield for substrate input in the tens of grams per liter. The ability to catalyze this non-natural C—C bond forming reaction in vivo expands the scope of transformations accessible to microbial organic synthesis and provides artificial metabolic pathways to complement nature's existing strategies for making cyclopropanes (L. A. Wessjohann et al., Chem. Rev. 103, 1625 (2003).

This concept has been demonstrated for a single P450 enzyme, from Bacillus megaterium, and for chimeras of the B. megaterium enzyme with other, related P450s from B. subtilis. Those of skill in the art, however, will recognize that other P450s from other organisms can be engineered to carry out cyclopropanation and ABC whole-cell catalysts can be made using those enzymes. In particular, the equivalent of the C400S mutation will improve the performance of other P450 enzymes for cyclopropanation and other carbene transfer reactions. One of skill in the art knows how to identify the equivalent residue to C400 in other P450s, based on sequence alignments, an example of which is given below. Methods known in the art, such as site-directed mutagenesis or gene synthesis, can be used to alter this residue to serine in any P450. If the resulting enzyme folds properly, it will serve as a catalyst for cyclopropanation. This mutation in a purified protein or whole cell catalyst will improve the activity over the parent enzyme that does not include this mutation.

For example, BLAST alignment (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the amino acid sequence of P450_(BM3) (CYP102A1) to other P450s, such as the one from Pseudomonas putida (CYP101A1, P450_(CAM)) or the mammalian enzyme from Oryctolagus cuniculus (CYP2B4), enables identification of the proximal cysteine residue or of the equivalent T268 (marked in bold), as shown below:

CYP102A1 380 ENPSAIPQH--------AFKPFGNGQRACIGQQFALHEATLVL 414 E  +A P H        +   FG+G   C+GQ  A  E  + L CYP101A1 329 ERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTL 371 CYP102A1 265 GHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRL 324 G +T    LSF++ FL K+P   Q+  E   R     +P+               E LR CYP101A1 249 GLDTVVNFLSFSMEFLAKSPEHRQELIERPER-----IPA------------ACEELLR- 290 CYP102A1 374  FRPERF--ENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFD 422 F P  F   N +      F PF  G+R C+G+  A  E  L    +L++F CYP2B4 408 FNPGHFLDANGALKRNEGFMPFSLGKRVCLGEGIARTELFLFFTTILQNFS 458 CYP102A1 257 QIITFLIAGHETTSGLLSFALYELVKNPHVLQKAAEEAARVL-VDPVPSYKQVKQLKYVG 315  +++   AG ETTS  L +    ++K PHV ++  +E  +V+     P+     ++ Y CYP2B4 291 TVLSLFFAGTETTSTTLRYGELLMLKYPHVTERVQKEIEQVIGSHRPPALDDRAKMPYTD 350

Therefore, the mutations C357S and T252A in CYP101A1 or C436S and T302A in CYP2B4 are expected to enhance the cyclopropanation activity in these enzymes. The mutation can be introduced into the target gene by using standard cloning techniques or by gene synthesis. The mutated gene can be expressed in the appropriate microbial host under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Cyclopropanation activity can be screened in vivo or in vitro by following product formation by GC or HPLC (see, Materials and Methods).

Because the ABC catalyst is genetically encoded and functions very well in whole cells, this catalyst can be incorporated into multi-enzyme pathways for biological synthesis in vivo, where multiple transformations of a substrate are carried out inside the cell. In particular embodiments, the EDA or other diazo reagent is provided exogenously to the medium and/or generated in situ.

The ability to extend the C400S mutation to other P450 scaffolds provides access to a variety of diazo compounds as carbenoid precursors. These include, but are not limited to, diazo esters (acceptor type), diazo β-keto ester or β-cyano esters (acceptor-aceptor type), and alkyl, aryl, or alkenyl substituted diazo esters (donor-acceptor) (FIG. 17A). These diazo compounds can be reacted inter- and intramolecularly with a variety of styrenes, aliphatic olefins, and allenes to provide biologically active compounds or organic building blocks for further reaction (FIG. 17B). For instance, P450 catalyzed reaction of a diazo ester with aryl or allylic silanes and boronic acids would form cyclopropanes that can then be used in Suzuki or Hiyama cross coupling reactions. Furthermore, cyclopropanation of geranylacetone at the C9 olefin by a trans selective P450 catalyst and EDA provides a key precursor to anthroplalone and noranthroplone, marine natural products that exhibit microgram cytotoxicity against B-16 melanoma cells (FIG. 17C). Lastly, treatment of 2,5-dimethylhexα-2,4-diene with a similar catalyst and EDA provides the ethyl ester of chrysanthemic acid, an important intermediate in the production of pyrethroid insecticides (FIG. 17D).

Materials and Methods

Unless otherwise noted, all chemicals and reagents for chemical reactions were obtained from commercial suppliers (Sigmα-Aldrich, Acros) and used without further purification. Silica gel chromatography purifications were carried out using AMD Silica Gel 60, 230-400 mesh. ¹H and ¹³C NMR spectra were recorded on either a Varian Mercury 300 spectrometer (300 MHz and 75 MHz, respectively), or a Varian Inova 500 MHz (500 MHz and 125 MHz, respectively), and are internally referenced to residual solvent peak. Data for ¹H NMR are reported in the conventional form: chemical shift (δppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling constant (Hz), integration. Data for ¹³C are reported in terms of chemical shift (δppm) and multiplicity. High-resolution mass spectra were obtained with a JEOL JMS-600H High Resolution Mass Spectrometer at the California Institute of Technology Mass Spectral Facility. Reactions were monitored using thin layer chromatography (Merck 60 silica gel plates) using an UV-lamp for visualization.

Gas chromatography (GC) analyses were carried out using a Shimadzu GC-17A gas chromatograph, a FID detector, and an Agilent J&W cyclosil-B column (30 m×0.25 mm, 0.25 μm film) and 2-phenylethanol as an internal standard. Injector temperature=300° C., oven temperature=130° C. for 30 min, pressure=175 kPa. Elution time: cis-cyclopropanes [19.7 min (2R,1S) and 21.0 min (2S,1R)], trans-cyclopropanes [25.8 min (2R,1R) and 26.4 min (2S,15)]. Cyclopropane product standards for the reaction of ethyl diazoacetate (EDA) with styrene (ethyl 2-phenylcyclopropane-1-carboxylate) and α-methylstyrene (ethyl 2-methyl-2-phenylcyclopropane-1-carboxylate) were prepared as reported (A. Penoni et al., Eur. J. Inorg. Chem. 2003, 1452 (2003)). These standards and enzyme-prepared cyclopropanes demonstrated identical retention times in gas chromatograms when co-injected, confirming product identity. Absolute stereoconfiguration of cyclopropane enantiomers was determined by measuring optical rotation of purified cyclopropane products from preparative bioconversion reactions using enantioselective BM3 variants and referenced to values taken from reference (N. Watanabe et al., Heterocycles 42, 537 (1996)). Authentic P450-catalyzed cyclopropane samples were also prepared as described herein and were characterized by NMR (¹H and ¹³C) and mass spectrometry.

Plasmids pCWori[BM3] and pET22 were used as cloning vectors. Site-directed mutagenesis was accomplished by standard overlap mutagenesis using primers bearing desired mutations (IDT, San Diego, Calif.). Electrocompetent Escherichia coli cells were prepared following the protocol of Sambrook et al., Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, New York, 1989), vol. 2. Restriction enzymes BamHI, EcoRI, XhoI, Phusion polymerase, and T4 ligase were purchased from New England Biolabs (NEB, Ipswich, Mass.). Alkaline phosphatase was obtained from Roche (Nutley, N.J.). The 1,000× trace metal mix used in expression cultures contained: 50 mM FeCl₃, 20 mM CaCl₂, 10 mM MnSO₄, 10 mM ZnSO₄, 2 mM CoSO₄, 2 mM CuCl₂, 2 mM NiCl₂, 2 mM Na₂MoO₄, and 2 mM H₃BO₃.

CO Binding Assay.

P450 concentration was determined from ferrous CO binding difference spectra using extinction coefficients of ε₄₅₀₋₄₉₀=91 mM⁻¹ cm⁻¹ for cysteine-ligated BM3 (T. Omura, R. Sato, J. Biol. Chem. 239, 2370 (1964)) and ε₄₁₁₋₄₉₀=103 mM⁻¹ cm⁻¹ for serine ligated ABC (K. P. Vatsis et al., J. Inorg. Biochem. 91, 542 (2002)).

P450 Expression and Purification.

For in vitro cyclopropanation reactions, BM3 variants were used in purified form. Enzyme batches were prepared as follows. One liter TB_(amp) was inoculated with an overnight culture (100 mL, LB_(amp)) of recombinant E. coli BL21(DE3) cells harboring a pCWori plasmid encoding the P450 variant under the control of the tac promoter. After 3.5 h of incubation at 37° C. and 250 rpm shaking (OD₆₀₀ ca. 1.8), the incubation temperature was reduced to 25° C. (30 min), and the cultures were induced by adding IPTG to a final concentration of 0.5 mM. The cultures were allowed to continue for another 24 hours at this temperature. After harvesting the cells by centrifugation (4° C., 15 min, 3,000×g), the cell pellet was stored at −20° C. until further use but at least for 2 h. The cell pellet was resuspended in 25 mM Tris.HCl buffer (pH 7.5 at 25° C.) and cells were lysed by sonication (2×1 min, output control 5, 50% duty cycle; Sonicator, Heat Systems—Ultrasonic, Inc.). Cell debris was removed by centrifugation for 20 min at 4° C. and 27,000×g and the supernatant was subjected to anion exchange chromatography on a Q Sepharose column (HiTrap™ Q HP, GE Healthcare, Piscataway, N.J.) using an AKTAxpress purifier FPLC system (GE healthcare). The P450 (or P411) was eluted from the Q column by running a gradient from 0 to 0.5 M NaCl over 10 column volumes (P450 elutes at 0.35 M NaCl). The P450 (or P411) fractions were collected and concentrated using a 30 kDa molecular weight cut-off centrifugal filter and buffer-exchanged with 0.1 M phosphate buffer (pH=8.0). The purified protein was flash-frozen on dry ice and stored at −20° C. P450 and P411 concentrations were determined in triplicate using the CO binding assay described above (10 μL P450 and 190 μL 0.1 M phosphate buffer, pH 8.0, per well).

For crystallization experiments, a two-step purification was performed using the AKTAxpress purifier FPLC system. Frozen cell pellets containing expressed, 6×His tagged heme domains were resuspended in Ni-NTA buffer A (25 mM Tris.HCl, 200 mM NaCl, 25 mM imidazole, pH 8.0, 0.5 mL/gcw) and lysed by sonication (2×1 min, output control 5, 50% duty cycle). The lysate was centrifuged at 27,000×g for 20 min at 4° C. to remove cell debris. The collected supernatant was first subjected to a Ni-NTA chromatography step using a Ni sepharose column (H isTrap-HP, GE healthcare, Piscataway, N.J.). The P450 (or P411) was eluted from the Ni sepharose column using 25 mM Tris.HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Ni-purified protein was buffer exchanged into 25 mM Tris.HCl pH 7.5 using a 30 kDa molecular weight cut-off centrifugal filter and subsequently loaded onto a Q sepharose column (HiTrap™ Q HP, GE healthcare, Piscataway, N.J.) and purified to homogeneity by anion exchange. The P450 (or P411) was eluted from the Q column by running a gradient from 0 to 0.5 M NaCl over 10 column volumes. P450 (or P411) fractions were collected and buffer exchanged into 25 mM Tris.HCl pH 7.5, 25 mM NaCl. The purified protein was concentrated with a 30 kDa molecular weight cut-off centrifugal filter to approximately 10 mg/mL. 50 μL aliquots were flash frozen on dry ice and stored at −80° C. until needed.

Protein Crystallography.

BM3-CIS and ABC-CIS were crystallized by vapor diffusion. A 1:1 mixture of protein stock (10 mg/mL in 25 mM Tris.HCl pH 7.5, 25 mM NaCl) and mother liquor was combined in 24 well sitting drop plates (Hampton Research). Optimal crystallization conditions for BM3-CIS were found in 0.1 M sodium cacadolyte, pH 5.7, 0.14 MgCl₂ and 17% PEG 3350. BM3-CIS crystals typically grew over a span of 7-14 days. ABC-CIS crystals optimally formed in 0.1 M Bis-Tris, pH 5.3, 0.2 M sodium formate and 18% PEG 3350. Initial ABC-CIS drops are marked with a dense layer of protein precipitate; however, after 36-48 hours, noticeable protein crystals were observed underneath the precipitate layer.

X-Ray Data Collection and Protein Structure Determination.

X-ray diffraction data were collected at the General Medical Sciences and Cancer Institutes Structural Biology Facility (GM/CA) at the Advanced Photon Source (APS, Argonne National Laboratory) using beamline ID23-D and a MAR300 CCD detector. Data were collected at 100K and a wavelength 0f 1.033 Å. Data collections statistics are listed in Table 38. Diffraction datasets were integrated with XDS (W. Kabsch, Acta Crystallogr. D66, 133 (2010)) and scaled using SCALA (P. Evans, Acta Crystallogr. D62, 72 (2006)). Initial phases were determined by molecular replacement against the closed form of wild type BM3_(heme) structure taken from PDB 1JPZ (D. C. Haines et al., Biochemistry 40, 13456 (2001)), chain B using MOLREP software (A. Vagin, A. Teplyakov, J. App. Crystallogr. 30, 1022 (1997)), a component of the CCP4 crystallography software suite (S. Bailey, Acta Crystallogr. D50, 760 (1994)). Refinement was accomplished by iterative cycles of manual model building within COOT (P. Emsley, K. Cowtan, Coot: Acta Crystallogr. D60, 2126 (2004)) and automated refinement using REFMAC (G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta Crystallogr. D53, 240 (1997)) within CCP4. Final cycles of REFMAC refinement included TLS parameters. Non-crystallographic symmetry constraints were not used during refinement. Model quality was assessed using the ‘complete validation’ tool inside of the PHENIX software suite (P. D. Adams et al., Acta Crystallogr. D66, 213 (2010)). Simulated annealing omit maps were also calculated using Phenix. Ramachandran outliers generally lie in poorly structured loops connecting BM3 F and G helices. These residues are often missing or marked by poor density in these and other BM3 structures within the protein database. All protein structure figures and alignments were generated using PyMol software (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.).

Thermostability Measurements.

Duplicate measurements were taken for all values reported in FIG. 30. Purified P450 (or P411) solutions (4 μM, 200 μL) were heated in a thermocycler (Eppendorf) over a range of temperatures (40° C.-70° C.) for 10 min followed by rapid cooling to 4° C. for 1 min. The precipitate was removed by centrifugation. The concentration of folded P450 (or P411) remaining in the supernatant was measured by CO-difference spectroscopy (as described above). The temperature at which half of the protein was denatured (T₅₀) was determined by fitting the data to the equation:

${f(T)} = \frac{100}{1 + ^{- {a{({\frac{1}{T} - \frac{1}{T_{50}}})}}}}$

(J. D. Bloom et al., Proc. Natl. Acad. Sci. U.S.A. 103, 5869 (2006)).

Typical Procedure for In Vitro Small-Scale Cyclopropanation Bioconversions Under Anaerobic Conditions.

Small-scale reactions (400 μL) were conducted in 2 mL crimp vials (Agilent Technologies, San Diego, Calif.). P450 solution (80 μL, 100 μM) was added to the vial with a small stir bar before crimp sealing with a silicone septum. Phosphate buffer (260 μL, 0.1 M, pH=8.0) and 40 μl, of a solution of the reductant (100 mM Na₂S₂O₄, or 20 mM NADPH) were combined in a larger crimp-sealed vial and degassed by bubbling argon through the solution for at least 5 min (Fig S1). In the meantime, the headspace of the 2 mL reaction vial with the P450 (or P411) solution was made anaerobic by flushing argon over the protein solution (with no bubbling). When multiple reactions were conducted in parallel, up to 8 reaction vials were degassed in series via cannulae. The buffer/reductant solution (300 μL) was syringed into the reaction vial, while under argon. The gas lines were disconnected from the reaction vial before placing the vials on a plate stirrer. A 40× styrene solution in MeOH (10 μL, typically 1.2 M) was added to the reaction vial via a glass syringe, and left to stir for about 30 s. A 40×EDA solution in MeOH was then added (10 μL, typically 400 mM) and the reaction was left stirring for the appropriate time. The final concentrations of the reagents were typically: 30 mM styrene, 10 mM EDA, 10 mM Na₂S₂O₄, 20 μM P450.

The reaction was quenched by adding 30 μL HCl (3M) via syringe to the sealed reaction vial. The vials were opened and 20 μL internal standard (20 mM 2-phenylethanol in MeOH) was added followed by 1 mL ethyl acetate. This mixture was transferred to a 1.8 mL eppendorf tube which was vortexed and centrifuged (16,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by chiral phase GC.

A slightly modified work-up was implemented for kinetic experiments. The reactions were quenched after the set time by syringing 1 mL EtOAc to the closed vials and immediately vortexing the mixture. The vials were then opened and 20 μL internal standard was added. The mixture was transferred to a 1.8 mL eppendorf tube, vortexed and centrifuged (16,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by GC.

Media and Cell Cultures for In Vivo Cyclopropanation.

E. coli [BL21(DE3))] cells were grown from glycerol stock overnight (37° C., 250 rpm) in 5 ml M9Y medium (1 L: 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 5.0 g NH₄C1, 0.24 g MgSO₄, 0.01 g CaCl₂, 1.5% yeast extract, 1 mL micronutrients, 0.1 mg mL⁻¹ ampicillin). The pre-culture was used to inoculate 45 mL of M9Y medium in a 125 mL Erlenmeyer flask and this culture was incubated at 37° C., 250 rpm for 2 h and 30 min. At OD₆₀₀=1.2, the cultures were cooled to 25° C. and the shaking was reduced to 160 rpm before inducing with IPTG (0.25 mM) and 6-aminolevulinic acid (0.25 mM). Cultures were harvested after 20 h and resuspended (OD₆₀₀=30) in nitrogen-free M9 medium (1 L: 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 0.24 g MgSO₄, 0.01 g CaCl₂, 1 mL micronutrients). The micronutrient solution contains 0.15 mM (NH₄)₆MO₇O₂₄, 20.0 mM H₃BO₃, 1.5 mM CoCl₂, 0.5 mM CuSO₄, 4.0 mM MnCl₂, and 0.5 mM ZnSO₄. Aliquots of the cell suspension were used for determination of the cell dry weight (cdw, 2 mL) and P450 (or P411) expression level (4 mL).

Small-Scale Whole-Cell Bioconversions.

E. coli cells (OD₆₀₀=30, 425 μL) were made anaerobic by bubbling argon through the cell suspension in a crimped 2 mL vial. A degassed solution of glucose (50 μL, 20 mM) was added to the cells before adding EDA (12.5 μl, of a 400 mM solution in MeOH) and olefin (12.5 μL of a 1.2 M solution in MeOH). The reactions were stirred at room temperature for the appropriate and were worked up by adding 20 μL of the internal standard (20 mM 2-phenylethanol) and extracting with 1 mL ethyl acetate. The organic layer was dried over Na₂SO₄ before analyzing the product mixture by chiral phase GC.

Preparative-Scale Whole-Cell Bioconversions.

E. coli [BL21(DE3)] cells were grown from glycerol stock overnight (37° C., 250 rpm) in 50 ml M9Y medium. The pre-culture was used to inoculate 2-475 mL of M9Y medium in 2-1 L Erlenmeyer flask (using 25 mL each) and this culture was incubated at 37° C., 250 rpm for 2 h and 30 min. At OD₆₀₀=1.8, the cultures were cooled to 25° C. and the shaking was reduced to 150 rpm before inducing with IPTG (0.25 mM) and δ-aminolevulinic acid (0.25 mM). Cultures were harvested after 24 h and resuspended (OD₆₀₀=75) in nitrogen-free M9 medium. Aliquots of the cell suspension were used for determination of the cell dry weight (cdw, 2 mL) and P450 (or P411) expression level (2 mL). E. coli cells (OD₆₀₀=70, 53.6 mL) were made anaerobic by bubbling argon through the cell suspension in a 500 mL sealed round bottom flask. A degassed solution of glucose (1.4 mL, 500 mM) was added to the cells before adding EDA (1.36 mL, 85% EDA in DCM as packaged by Sigma Aldrich) and styrene (2.5 mL, neat). The reaction was stirred at room temperature under positive argon pressure for 24 h. The crude mixture was poured into 3-50 mL conical tubes and the reaction was quenched by the addition of HCl (1 mL, 3 M) to each tube. The aqueous mixtures were extracted with 1:1 EtOAc:hexanes (20 mL each) and centrifuged (5000 rpm, 5 min). The organics were collected and this extraction sequence was performed two more times. The organics were combined, dried over Na₂SO₄ then concentrated. Excess styrene was removed via azeotrope with H₂O/benzene and 1.85 g of crude product was isolated. Cis/trans selectivity of the reaction was determined via gas chromatography of this crude mixture. Column chromatography of the crude product with 8% Et₂O/hexanes afforded the desired products as a mixture of cis and trans isomers (1.63 g combined, 78% yield). Based on comparison of crude and purified yields, the crude product was approximately 88% pure. NMR of the isolated products were identical to those reported in the literature (P. S. Coelho et al., Science 339, 307 (2013)).

Time Course of In Vivo and In Vitro Reactions.

Following the procedure for small scale bioconversions, a series of in vivo and in vitro reactions were set up and EDA was added to each sample at time 0 hours. Time points were taken at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 hours. Each reported yield reflects an average of two independent reactions that were allowed to stir for the indicated amount of time. The error bars shown reflect the two unaveraged data points. Yields of each reaction were determined by GC.

Potentiometric Titrations.

Enzyme samples were buffer-exchanged into 100 mM KPO₄, 100 mM KCl, pH 7.4, and deoxygenated via 4×20 gentle pump-backfill cycles with argon, with care taken to avoid bubbling. Potentiometric redox titrations were performed in an anaerobic glove box, using a quartz spectroelectrochemical cell with path length of 1 mm, platinum mesh working electrode, platinum wire counter electrode, and a Ag/AgCl electrode (Bioanalytical Systems, Inc.) was used as the reference (Ag/AgCl vs NHE: +197 mV). Protein solutions consisted of approximately 600 μL of 50-100 μM protein with the following mediators added to ensure electrochemical communication between the protein and electrode: methyl viologen (5 μM), benzyl viologen (10 μM) and 2-hydroxy-1,4-napthaquinone (20 μM). Enzyme samples were titrated using sodium dithionite (reduction) and potassium ferricyanide (re-oxidation). The open circuit potential of the cell was monitored (WaveNow potentiostat, Pine Research Instrumentation) over a 10 minute equilibration period, and spectra were recorded using a Ocean Optics spectrometer (USB2000+). The reduction potentials (E^(o)′) were determined by fitting the data to the one-electron Nernst equation.

Summary of P450-Derived Cyclopropanation Catalysts.

Mutations in cyclopropanation catalysts are reported with respect to wild-type BM3. The heme domain comprises the first 462 amino acids in the BM3 sequence.

-   ABC: BM3+C400S -   BM3-CIS: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A     A290V, L353V, I366V, E442K -   ABC-CIS: BM3-CIS+C400S

Rationale for the C400S Mutation.

The very negative Fe^(III/II) potential of cytochromes P450 relative to other heme proteins has been ascribed to the strong donating character of the axial cysteinate ligand. This effect has been modeled in cytochrome c: substitution of the native axial methionine for cysteine decreases the Fe^(III/II) potential by an impressive 652 mV, from 262 mV vs SHE for the Met/His ligated variant to −390 mV vs SHE for the Cys/His variant (A. L. Raphael, H. B. Gray, J. Am. Chem. Soc. 113, 1038 (1991)). Even within cytochrome P450, the reduction potential can be further reduced by increasing the electron donating character of the cysteinate ligand. Removal of a single amide proton proposed to stabilize the cysteinate negative charge shifts the Fe^(III/II) potential negative by 35-45 mV with respect to wild-type (S. Yoshioka et al., J. Am. Chem. Soc. 124, 14571 (2002)).

In order to facilitate cyclopropanation activity in vivo, it was necessary to shift the reduction potential sufficiently positive to allow reduction by NADPH. In cytochrome c, it was observed that axial ligation by a weakly donating water molecule raises the reduction potential of the His/H₂O ligated variant (Fe^(III/II): −45 mV vs SHE) by 345 mV compared to the Cys/His variant (A. L. Raphael, H. B. Gray, J. Am. Chem. Soc. 113, 1038 (1991)). To that end, it was hypothesized that substitution of the P450 cysteinate axial ligand with the weakly donating serine alcohol would shift the C400S reduction potential positive compared to wild-type. The pK_(a) of serine (˜15) is approximately 7 pH units above that of cysteine (˜8), and so while cysteine remains deprotonated as the cysteinate ligand in both ferric and ferrous states of the enzyme, serine would remain protonated in at least the ferrous form. The serine-ligated mammalian P450 mutant has been suggested to be serinate in the ferric form, and serine in the ferrous form based on analysis of absorption spectra and magnetic circular dichroism (R. Perera et al., Arch. Biochem. Biophys. 507, 119 (2011)), and it was hypothesized that similar ligation in Ser-P450-BM3 would result in a more positive Fe^(III/II) potential.

Supplementary Data Physical Characterization of the C400S Mutant

X-Ray Crystallography Statistics.

To confirm heme coordination by an axial C400S mutation and to investigate the nature of enhanced stereoselectivity observed in ABC-CIS, crystal structures of both proteins were determined (Table 38) to assess any structural changes that may have occurred due to the axial Cys→Ser mutation. The top panels of FIG. 9 shows alignments of BM3-CIS (green) and ABC-CIS (peach) with left, middle and right panels showing active site residues, the active site I-helix, and global protein fold, respectively. No significant structural changes were observed (RMSD 0.52 Å). FIG. 9, middle panels: Large variations are observed upon comparing BM3-CIS with the open (ligand-free) form of wild type BM3 (purple, taken from PDB#21J2, RMSD 1.2 Å). Pronounced rearrangements are observed in active site side chain residues (left) as well as rotations within the I-helix. Global movements are also observed in the N-terminal beta domain as well as F- and G-helices (right, marked by double headed arrows). These movements are consistent with well-known transitions that occur upon substrate binding and are important for native monooxygenation catalysis. FIG. 9, bottom panels: Alignment of BM3-CIS with a ligand-bound BM3 structure (cyan, taken from PDB#1JPZ, RMSD 0.52 Å) demonstrates that BM3-CIS and ABC-CIS mimic the closed protein conformation even in the absence of substrate. Protein aligments were carried out using the align tool of PyMol (PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC.).

UV-Vis Spectroscopy

FIG. 21 illustrates the absolute spectra for ferric (blue), dithionite reduced ferrous (red) and carbon monoxide bound ferrous (green) ABC-CIS_(heme). Soret bands (nm): Fe^(III), 404; Fe^(II), 410, 425; Fe^(II)-CO, 411. Fe^(II)-CO displays α and β bands at 532 and 565 nm. Insert shows the carbon monoxide ferrous (pink) and the dithionite reduced ferrous (yellow) enzymes at 4.5 μM protein concentration. The shoulder at 410 nm for the ferrous spectrum is due to incomplete reduction to FeII under the aerobic conditions in which these spectra were taken. Reduction of cytochrome P411 under strict anaerobic conditions, as is the case for the redox titrations (FIGS. 25 and 27) gives a single peak at 421 nm.

FIG. 22 illustrates the absolute spectra for ferric (blue), dithionite reduced ferrous (red) and carbon monoxide bound ferrous (green) ABC-CIS_(holo). Soret bands (nm): Fe^(III), 404; Fe^(II), 410, 422; Fe^(II)-CO, 411. Fe^(II)-CO displays α and β bands at 533 and 566 nm. Ferric spectrum displays a broad peak at 465 nm.

FIG. 23 illustrates the difference spectra for ferrous carbonyl with respect to ferrous for: (A) ABC-CIS_(heme) and (B) ABC-CIS_(holo).

Redox Titrations

FIG. 24 illustrates the potentiometric redox titration for P450_(BM3-heme) with overlaid Nernst curve fit to E^(o)′=−420 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

FIG. 25 illustrates the potentiometric redox titration for P411_(BM3-heme) with overlaid Nernst curve fit to E^(o)′=−293 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

FIG. 26 illustrates the potentiometric redox titration for P450_(BM3-heme)-CIS with overlaid Nernst curve fit to E^(o)′=−360 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

FIG. 27 illustrates the potentiometric redox titration for P411_(BM3-heme)-CIS with overlaid Nernst curve fit to E^(o)′=−265 mV. Inset shows spectral changes upon each sub-stoichiometric addition of sodium dithionite (dashed line: fully ferric; solid line: fully ferrous).

In vitro Cyclopropanation Activities of ABC-CIS and ABC

Michaelis-Menten Kinetics

Determination of Initial Rates.

Both styrene and EDA concentrations were varied in the presence of the enzymes expressed as the heme-domain (0.5 or 1.0 μM BM3-CIS_(heme)). Reactions were set up in phosphate buffer (pH=8.0) with Na₂S₂O₄ as the reductant at 298 K, and were worked-up as described above. Three time points were taken and used to determine the rate of product formation by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=100° C. 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution time: cis-cyclopropanes (19.20 min and 19.33 min), trans-cyclopropanes (20.44 min). Kinetic parameters were determined by fitting the data to the standard Michaelis-Menten model.

Enhanced Cis Selectivity and Substrate Scope

FIG. 28 illustrates the activity under anaerobic vs. aerobic conditions with dithionite for variant 9-10A-TS-F87V-T268A (also called BM3-CIS or P450_(BM3-heme)-CIS) and BM3-CIS-C400S (also called ABC-CIS or P411_(BM3-heme)-CIS).

Small-Scale Reactions.

Selected P450 catalysts were surveyed at a small-scale reaction (400 μL total volume) for each combination of reagents (olefins and diazo esters). The small-scale anaerobic bioconversions were conducted as described above and were analyzed by GC.

GC methods for these products are reported in reference (P. S. Coelho et al Science 339, 307 (2013)). Table 41 shows the enhanced Z selectivity for ABC-CIS (P411_(BM3)-CIS) over BM3-CIS (P450_(BM3)-CIS).

Monooxygenation vs. Cyclopropanation Activities for BM3-CIS and ABC-CIS

Activity Under Anaerobic Vs. Aerobic Conditions with NADPH as the Reductant.

Small-scale reactions (400 μL total volume) were conducted as described above with the following modifications: glucose dehydrogenase (GDH, 4 μL, 225 U mL⁻¹) was added to the reaction vial together with the P450 solution. Glucose (40 μL, 250 mM) and NADPH (40 μL, 5 mM) were degassed together with the buffer solution. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Table 36 illustrates in vitro activities for purified P411_(BM3)-CIS vs P450_(BM3)-CIS driven by NADPH.

Choice of Reductant: NADPH vs NADH

Table 42 illustrates in vitro ABC-CIS_(holo) cyclopropanation driven by Na₂S₂O₄, NADPH and NADH.

In Vitro Cyclopropanation Activities of BM3 and ABC

Small-scale (500 μL) reactions were conducted as described above with purified BM3 and ABC catalysts. Table 44 illustrates in vitro cyclopropanation activities of BM3 and ABC driven by NADH.

Whole-Cell Cyclopropanation Catalysts

All experiments using whole-cells were done in triplicate; the error bars represent the standard deviation of the measurements. ‘Total turnovers’ is defined herein as the amount of cyclopropane product (mmol) formed per mass of catalyst (g_(cdw)).

Effect of Glucose Addition

FIG. 29 illustrates the effect of adding exogenous glucose (2 mM) on olefin cyclopropanation catalyzed by E. coli whole cells expressing 9-10A-TS-F87V-T268A (also called BM3-CIS and P450_(BM3)-CIS) or BM3-CIS-C400S (also called ABC-CIS and P411_(BM3)-CIS).

Effect of C400S on Thermostability

FIG. 30 illustrates thermostabilities of heme domains of BM3-CIS (P450_(BM3)-CIS in blue) and ABC-CIS (P411_(BM3)-CIS in red). The C400S mutation stabilizes the heme domain by +1.7° C. The T₅₀ is the temperature at which half of the enzyme population has unfolded. Error bars correspond to 1-σ (68.3%) confidence intervals for the T₅₀.

Anaerobic vs Aerobic Reaction Conditions

FIG. 31 illustrates the effect of dioxygen exposure on whole-cell catalyzed cyclopropanation. ABC-CIS (P411_(BM3)-CIS) is strongly inhibited by dioxygen in vivo. All reactions had a cell density equivalent to OD₆₀₀=25. Reactions were conducted in the absence of exogenous glucose. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Product formation is defined as the amount of cyclopropane product (mmol) formed per mass of catalyst (g_(cdw)).

Empty Plasmid, No Induction Controls and Dithionite Addition to Whole-Cells

FIG. 32 illustrates empty plasmid, no-induction controls and dithionite addition to whole cells. E. coli cells carrying the ABC-CIS (P411_(BM3)-CIS) gene but grown without the addition of IPTG (ABC-CIS no induction); E. coli cells carrying the pcWori plasmid but not the ABC-CIS gene (empty pcWori); ABC-CIS reaction with the addition of exogenous dithionite instead of glucose (ABC-CIS+dithionite). Reactions were left for two hours at 298 K. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Product formation is defined as the amount of cyclopropane product (mmol) formed per mass of catalyst (g_(cdw)).

Effect of Cell Density

FIG. 33 illustrates that increasing cell density increases cyclopropane yields up ˜80%. Total turnovers do not increase for cell densities higher than OD₆₀₀=20. Measurements were taken in triplicate and the error bars represent the standard deviation from the mean value. Product formation is defined as the amount of cyclopropane product (mmol) formed per mass of catalyst (g_(cdw)).

Effect of Styrene Concentration

FIG. 14 illustrates the effect of using 1, 2, 3, 4 and 5 equivalents of styrene on reaction yield. Excess styrene gives only small improvements in yield.

Lysate Compared to Intact Whole-Cells

Table 45 illustrates lysate activity compared to in vivo activity.

Lyophilization of Whole-Cell Catalysts

Cells were lyophilized in 10% sucrose (m/V) and were stored at 4° C. for two weeks. An appropriate mass of the resulting powder was transferred to a 2 mL glass vial, which was crimp sealed and purged with argon. Degassed solutions of nitrogen-free M9 medium and glucose (20 mM) were added via syringe. Cells were resuspended to OD₆₀₀=25 and 2 mM final concentration of glucose.

Table 47 illustrates the cyclopropanation activity of lyophilized ABC-CIS whole-cell catalysts.

Example 4 A Serine-Substituted P450 Catalyzes Highly Efficient Carbene Transfer to Olefins In Vivo

Genetically encoded catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. This example illustrates the design of a unique serine-heme ligated cytochrome “P411” that catalyzes efficient and selective carbene transfers from diazoesters to olefins in intact Escherichia coli cells. The mutation C400S in cytochrome P450_(BM3) gives a signature ferrous-CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting state Fe^(III/II) reduction potential, and significantly improves NAD(P)H-driven cyclopropanation activity.

Introduction

Genetically programmed whole-cell biocatalysts are readily produced in simple growth media, do not require further purification or isolation and can be engineered with biosynthetic pathways for the elaboration of complex molecules (Ajikumar, P. K. et al. Science 330, 70-74 (2010); Westfall, P. J. et al. Proc. Natl. Acad. Sci. U.S.A. 109, E111-E118 (2012); Kataoka, M. et al. Appl. Microbiol. Biotechnol. 62, 437-445 (2003)). The range of accessible transformations, however, is currently limited to the chemical repertoire of natural enzymes. Designing enzymes for non-natural reactions in vivo has been challenging due to the requirements for assembly of the functional catalyst, the compatibility of synthetic reagents in the cellular milieu, and cell permeability to allow substrate influx and product release. The catalysis of non-natural transformations inside cells will enable alternative biosynthetic routes to natural and artificial products, biocatalytic production of chemicals currently made using synthetic reactions, and will expand the chemical toolbox available for in vivo studies of cellular function (Boyce, M. & Bertozzi, C. R. Nature Methods 8, 638-642 (2011)).

The preceding examples demonstrate that a few amino acid mutations in a bacterial cytochrome P450 monooxygenase can unlock significant cyclopropanation activity in vitro. Variants of P450_(BM3) from Bacillus megaterium catalyze hundreds of turnovers of formal carbene transfers from diazoesters (e.g., ethyl diazoacetate, EDA) to olefins (e.g., styrene) in the presence of a reductant, forming cyclopropane products with high levels of diastereoselectivity and enantioselectivity (Coelho, P. S. et al. Science 339, 307-310 (2013)). Olefin cyclopropanation is widely used in the synthesis of fine chemicals (Lebel, H. et al. Chem. Rev. 103, 977-1050 (2003)), and state-of-the-art asymmetric organometallic catalysts are able to catalyze thousands to tens of thousands of turnovers (Evans, D. A. et al. J. Am. Chem. Soc. 113, 726-728 (1991); Davies, H. M. L. & Venkataramani, C. Org. Lett. 5, 1403-1406 (2003); Maas, G. Chem. Soc. Rev. 33, 183-190 (2004)). Because P450_(BM3) variants are readily expressed in functional form and can catalyze non-natural carbene transfers without requiring artificial cofactors or posttranslational modifications, this system may be suitable for in vivo catalysis. To initiate the catalytic cycle inside a cell, it is necessary to reduce the enzyme to the catalytically active ferrous-P450 with an endogenous reducing agent such as NAD(P)H. Based on consideration of heme ligation control of the P450 Fe^(III)/Fe^(II) reduction potential, genetically encoded cytochrome P411 enzymes have been designed which catalyze efficient and selective olefin cyclopropanation in intact cells.

Results

Cytochrome P450_(BM3)-catalyzed cyclopropanations require substoichiometric (with respect to diazoester and olefin) reductant and proceed optimally under anaerobic conditions (Coelho, P. S. et al. Science 339, 307-310 (2013)). This suggests that diazoester activation and carbene transfer involve a reduced P450-bound Fe^(II)-heme prosthetic group as opposed to the resting state Fe^(III)-heme (FIG. 35A). Active cyclopropanation catalysts derived from either full-length P450_(BM3), which contains a catalytic heme domain fused to a NADPH-driven P450-reductase domain, or the isolated heme domain (P450_(BM3-heme)) show marked preference for strong reducing agents such as dithionite (E^(o)′=−660 mV, all potentials vs SHE) over native NAD(P)H (E^(o)′=−320 mV) (Coelho, P. S. et al. Science 339, 307-310 (2013)). Reduced activity in the presence of NAD(P)H suggests a limited substrate-induced low-spin (E^(o)′ Fe^(III/II)=−430 mV) to high-spin (E^(o)′ Fe^(III/II)=−290 mV) transition of the P450 heme-iron (Ost, T. W. B. et al. Biochemistry 40, 13421-13429 (2001)), which, while essential for monooxygenation, may not be achievable in this engineered system due to the poor affinity for the non-natural substrates (K_(M)˜5 mM) (Coelho, P. S. et al. Science 339, 307-310 (2013)). It was hypothesized that raising the reduction potential of the resting state enzyme to facilitate NAD(P)H-driven reduction would enhance Fe^(II) catalysis in vivo. Aware that the reduction potential of heme proteins can be tuned by axial ligand mutations (Wuttke, D. S. & Gray, H. B. Curr. Opin. Struct. Biol. 3, 555-563 (1993); Reedy, C. J. et al. Nucleic Acids Research 36, D307-D313 (2008)), it was reasoned that substituting the axial cysteine thiolate in P450_(BM3) with the weakly donating serine alcohol should raise the Fe^(III/II) potential (FIG. 35A). Furthermore, axial cysteinate ligation is essential for dioxygen activation and stabilization of the active ferryl-porphyrin cation radical oxidant (FIG. 1, compound I) during monooxygenation (Dawson, J. H. Science 240, 433-439 (1988)), and axial cysteine to serine substitutions have been reported to abolish monooxygenation activity in mammalian P450s (Vatsis, K. P. et al. J Inorg. Biochem. 91, 542-553 (2002)). Because free hemin is also a (poor) cyclopropanation catalyst (Coelho, P. S. et al. Science 339, 307-310 (2013)), an axial cysteine to serine mutation (C400S in P450_(BM3)) would maintain carbene transfer activity while eliminating monooxygenation activity.

The C400S mutation was introduced into a cis-selective cyclopropanation catalyst from the preceding examples, P450_(BM3)-CIS (13 mutations from P450_(BM3)), to contrast with the trans-selectivity observed with iron-porphyrins (Wolf, J. R. et al. J. Am. Chem. Soc. 117, 9194-9199 (1995)). P450_(BM3)-CIS catalyzes hundreds of turnovers in the presence of dithionite in vitro and forms the ethyl 2-phenylcyclopropane-1-carboxylate product with 71% cis-selectivity and −94% enantiomeric excess (ee_(cis)) (Coelho, P. S. et al. Science 339, 307-310 (2013)). UV-vis spectra for the green-brown P450_(BM3)-CIS-C400S (P411_(BM3-CIS), FIGS. 21-23), marked by a ferrous carbon monoxide-bound complex at 411 nm, were consistent with those reported for a Ser-ligated mammalian P450 (Vatsis, K. P. et al. J Inorg. Biochem. 91, 542-553 (2002); Perera, R. et al. Arch. Bioch. Bioph. 507, 119-125 (2011)) (Table 39). Because of this signature Fe^(II)-CO band at 411 nm, these new P450-derived Ser-ligated enzymes cytochrome are called “P411s”. The C400S P450_(BM3) variants will hereafter be referred to as “P411_(BM3)” enzymes. Heme-serine ligation in P411_(BM3)-CIS was further confirmed by determining the crystal structures of the P450_(BM3)-CIS and P411_(BM3)-CIS heme domains at 2.5 and 3.3 Å, respectively (FIG. 9 and Table 38; PDB: 4H23 and 4H24); the structures were superimposable (RMSD=0.52 Å; FIG. 9). Despite the limited resolution of the P411_(BM3-CIS) structure, simulated annealing omit maps generated in the absence of modeled heme and C400S showed density consistent with heme coordination by a proximal amino acid side chain (FIG. 35B and FIG. 20).

Potentiometric redox titrations were performed using the truncated heme domains of wild-type P450_(BM3), its C400S variant (P411_(BM3)), P450_(BM3)-CIS and P411_(BM3)-CIS (FIGS. 24-27). The C400S mutation raised the reduction potential of the resting state wild-type P450_(BM3-heme) by +127 mV (E^(o)′ Fe^(III/II) _(ser)=−293 mV for P411_(BM3-heme)), a shift similar in magnitude to that which occurs in P450_(BM3) upon substrate binding (Ost, T. W. B. et al. Biochemistry 40, 13421-13429 (2001)). The 13 amino acid mutations in P450_(BM3-heme)-CIS increased the reduction potential by +60 mV with respect to wild-type P450_(BM3-heme) (E^(o)′ Fe^(III/II) _(cys)=−360 mV for P450_(BM3-heme)-CIS), but still left P450_(BM3-heme)-CIS with a lower reduction potential than NAD(P)⁺/NAD(P)H. Introducing the C400S mutation in P450_(BM3-heme)-CIS raised its reduction potential by another +95 mV (E^(o)′ Fe^(III/II) _(ser)=−265 mV for P411_(BM3-heme)-CIS). That the two C400S enzymes have resting state reduction potentials more positive than that of NAD(P)⁺/NAD(P)H should allow full-length P411s to be reduced by NAD(P)H even in the absence of substrate.

The truncated P411_(BM3-heme)-CIS was an active dithionite-driven cyclopropanation catalyst in vitro, with Michaelis-Menten parameters (k_(cat)=82 min⁻¹, K_(M-styrene)=4.6 mM, K_(M-EDA)=5.7 mM, FIG. 8), comparable to those of P450_(BM3-heme)-CIS (Table 40). P411_(BM3-heme)-CIS displayed considerably improved diastereo-(cis:trans 93:7) and enantioselectivity (−99% ee_(cis)) compared to its cysteine homologue (FIG. 28), an unexpected result given the similar active site geometries of the two catalysts (FIG. 9). For a variety of styrenyl substrates, P411_(BM3-heme)-CIS showed superior cis-selectivity relative to P450_(BM3-heme)-CIS (Table 41). The full-length, reductase-fused P411_(BM3)-CIS showed increased activity compared to holo P450_(BM3)-CIS when NADPH was used as the reductant under anaerobic conditions (FIG. 35C and Table 36). P450_(BM3)-CIS only formed small amounts of cyclopropanes when NADPH was used, and formed styrene oxide, via monooxygenation, as the major product under aerobic conditions. In contrast, P411_(BM3-CIS) produced negligible amounts of styrene oxide, confirming removal of monooxygenase activity, and was still able to form cyclopropanes under aerobic conditions, albeit with lower yields (43 TTN) due to oxygen inhibition (FIG. 35C). Dioxygen inhibition could be due to a two-electron oxidase activity as reported for CYP2B4-C436S (Vatsis, K. P. et al. J. Inorg. Biochem. 91, 542-553 (2002)). NADH drove P411_(BM3)-CIS-mediated cyclopropanation as efficiently as NADPH (Table 42), indicating that P411_(BM3-CIS) should be well suited for in vivo catalysis under anaerobic conditions where NADPH biosynthesis in E. coli does not take place. The apparent lack of the substoichiometric cofactor preference for cyclopropanation TTN contrasts with P450_(BM3)'s reported specificity for NADPH (Dunford, A. J. et al. Biochim. Bioph. Acta, Proteins and Proteomics 1794, 1181-1189 (2009)).

The efficiency of cyclopropanation using resting Escherichia coli [BL21(DE3)] cells grown in M9Y media (M9, 1.5% yeast extract) expressing full-length P450_(BM3)-CIS and P411_(BM3)-CIS was next investigated. Addition of glucose under anaerobic conditions significantly increased product yield (FIG. 29), presumably due to enhanced intracellular production of NADH, although other cellular reductants could also be involved in reducing the enzyme in vivo. P411_(BM3-CIS) catalyzed thousands of turnovers in vivo, was about four times more active than P450_(BM3)-CIS in the whole-cell system, and provided the cyclopropane products with enhanced cis-enantioselectivity (Table 43, entries 1 and 2). The C400S mutation compromises protein expression such that P411_(BM3-CIS) accounts for 2% of dry cell mass compared to 6% for BM3-CIS (entries 1 and 2). The reduced expression was not due to decreased protein stability, as C400S contributed to increased thermostability in the purified P411_(BM3)-CIS heme domain (P411_(BM3-heme)-CIS, FIG. 10). Full-length P411_(BM3)-CIS (entry 2), which contains both heme and diflavin reductase domains, was over two times more active on a molar basis than the truncated heme domain (P411_(BM3-heme)-CIS, entry 3), confirming that reduction to the ferrous state in vivo was important, but also showing that the reducing intracellular environment achieved heme reduction even in the absence of the reductase domain. The single C400S mutation in P411_(BM3) (entry 4) improved the in vivo cyclopropanation activity of wild-type P450_(BM3) (entry 5) by over two orders of magnitude. Purified full-length P411_(BM3) was also an efficient NADH-driven cyclopropanation catalyst in vitro, whereas P450_(BM3) was barely active (Table 44).

Both P411_(BM3)-CIS and P450_(BM3)-CIS whole cells were significantly inhibited by dioxygen (FIG. 31). Whole cells containing the P411_(BM3)-CIS gene but with no induction and whole cells devoid of the P411_(BM3-CIS) gene were able to form small amounts of cyclopropanes, but did so with stereoselectivity similar to that of free hemin (FIG. 32). This is not surprising since free hemin and other heme proteins present in cells are also able to catalyze styrene cyclopropanation at low levels (Coelho, P. S. et al. Science 339, 307-310 (2013)). Whole-cell P411_(BM3)-CIS catalysts were as stereoselective as purified P411_(BM3)-CIS in vitro at equivalent catalyst loading (vide infra), demonstrating that the overexpressed P411 enzyme outcompeted background catalysis. In vivo cyclopropanation was strongly inhibited by carbon monoxide (Table 43, entry 6), which irreversibly binds ferrous heme, confirming that catalysis occurs in the enzyme active site. Yield could be increased to 80% by increasing the cell density up to OD₆₀₀=50 (FIG. 33). Using excess styrene only slightly improved reaction yield (FIG. 14). Lysate of cells expressing full-length P411_(BM3)-CIS that were supplemented with NADH retained only about 30% of the activity of the intact whole cells and were not active in the absence of exogenous reductant (Table 45). Addition of dithionite inhibited P411_(BM3)-CIS whole-cell reactions and was less efficient than NADH in driving the reaction in cell lysate (FIG. 32 and Table 45).

In order to provide a direct comparison of full-length P411_(BM3-CIS) activity in vivo versus in vitro, both reactions were monitored at the same enzyme concentration over 8 hours (FIG. 34). On a molar basis, the in vivo catalyst showed almost 6 times higher TTN than the purified enzyme after 6 hours and retained the same stereoselectivity (75:25 cis:trans, −95% ee_(cis)). Both catalysts remained active over this period, indicating that the observed differences in yield and TTN are due to improved activity rather than enhanced catalyst stability in vivo. Gradual addition of EDA did not improve the reaction yield.

At high substrate loading (170 mM EDA, 400 mM styrene, added as neat reagents), more than 60,000 catalytic turnovers were observed in the in vivo reaction with P411_(BM3)-CIS (Table 43, entry 7). P411_(BM3)-CIS whole-cell reactions were readily scalable to make gram quantities of cyclopropanes with high stereoselectivity, product titer (27 g L⁻¹) and yield (78%, entry 8). No organic cosolvent was necessary, and the cyclopropane products were readily obtained by extraction with organic solvent at the end of the reaction. Furthermore, the cells could be lyophilized with a cryoprotectant such as sucrose and stored as a powder for weeks at 4° C. without degradation of catalytic activity or diastereo- and enantioselectivity (Table 47). Lyophilized cells can be readily packaged and distributed. These features render whole-cell P411 catalysts attractive for facile benchtop synthesis.

Cytochrome P411s are spectroscopically, electrochemically, and catalytically distinct from cytochrome P450s, providing a scaffold for engineering orthogonal heme-enzyme catalysis. Whole-cell catalysts based on serine-heme ligated P411s are easy to use and deliver enzymatic cyclopropanation with high conversion, optical purity and yield for substrate input in the tens of grams per liter. The ability to catalyze this non-natural C—C bond forming reaction in vivo expands the scope of transformations accessible to microbial organic synthesis and provides artificial metabolic pathways to complement nature's existing strategies for making cyclopropanes (Wessjohann, L. A. et al. Chem. Rev. 103, 1625-1647 (2003)).

Materials and Methods

All reagents were obtained from commercial suppliers (Sigmα-Aldrich) and used without further purification. ¹H and ¹³C NMR spectra were recorded on either a Varian Mercury 300 spectrometer (300 MHz and 75 MHz, respectively), or a Varian Inova 500 MHz (500 MHz and 125 MHz, respectively), and are internally referenced to residual solvent peak. High-resolution mass spectra were obtained with a JEOL JMS-600H High Resolution Mass Spectrometer. Gas chromatography (GC) analyses were carried out using a GC-17A gas chromatograph (Shimadzu), a FID detector, and a J&W cyclosil-B column (30 m×0.25 mm, 0.25 μm film, Agilent) and 2-phenylethanol as an internal standard. Injector temperature=300° C., oven temperature=130° C. for 30 min, pressure=175 kPa. Elution time: cis-cyclopropanes [19.7 min (2R,1S) and 21.0 min (2S,1R)], trans-cyclopropanes [25.8 min (2R,1R) and 26.4 min (2S,1S)]. Cyclopropane product standards for the reaction of ethyl diazoacetate (EDA) with styrene (ethyl 2-phenylcyclopropane-1-carboxylate) and α-methylstyrene (ethyl 2-methyl-2-phenylcyclopropane-1-carboxylate) were prepared as reported (Penoni, A. et al. Eur. J. Inorg. Chem., 1452-1460 (2003)). These standards and enzyme-prepared cyclopropanes demonstrated identical retention times in gas chromatograms when co-injected, confirming product identity. Absolute stereoconfiguration of cyclopropane enantiomers was determined by measuring optical rotation of purified cyclopropane products from preparative bioconversion reactions using enantioselective P450_(BM3) variants and referenced to values taken from reference (Watanabe, N. et al. Heterocycles 42, 537-542 (1996)). Authentic P450-catalyzed cyclopropane samples were also prepared and were characterized by NMR (¹H and ¹³C) and mass spectrometry, which matched literature values.

Plasmids pCWori[P450_(BM3)] and pET22 were used as cloning vectors. The C400S mutation was introduced by standard overlap mutagenesis using primers bearing the desired mutation (IDT, San Diego, Calif.).

Forward Primers: HF1: CAGGAAACAGGATCAGCTTACTCCCC BM3_C400S_F_nheI: GAAACGGTCAGCGTGCTAGCATCGGTCAGCAGTTCG Respective Reverse Primers: BM3_C400S_R_nheI: CGAACTGCTGACCGATGCTAGCACGCTGACCGTTTC pCWori-Rev: GCGTATCACGAGGCCCTTTCGTCTTCAAGC

Electrocompetent Escherichia coli cells were prepared following the protocol of Sambrook et al., Molecular cloning: a laboratory manual. Vol. 2 (Cold Spring Harbor Laboratory Press, New York, 1989)). Restriction enzymes BamHI, EcoRI, XhoI, Phusion polymerase, and T4 ligase were purchased from New England Biolabs (NEB, Ipswich, Mass.). Alkaline phosphatase was obtained from Roche (Nutley, N.J.). The 1,000× trace metal mix used in expression cultures contained 50 mM FeCl₃, 20 mM CaCl₂, 10 mM MnSO₄, 10 mM ZnSO₄, 2 mM CoSO₄, 2 mM CuCl₂, 2 mM NiCl₂, 2 mM Na₂MoO₄, and 2 mM H₃BO₃.

CO Binding Assay.

P450 concentration was determined from ferrous CO binding difference spectra using extinction coefficients of ε₄₅₀₋₄₉₀=91 mM⁻¹ cm⁻¹ for cysteine-ligated P450_(BM3) (Omura, T. & Sato, R. J. Biol. Chem. 239, 2370-2378 (1964)) and ε₄₁₁₋₄₉₀=103 mM⁻¹ cm⁻¹ for serine ligated P411_(BM3) (Vatsis, K. P. et al. J. Inorg. Biochem. 91, 542-553 (2002)). The in vivo P450 (or P411) concentration was determined by conducting the CO assay in the lysate of an aliquot of cells in the same cell density as used for the whole-cell reactions.

P450 Expression and Purification.

For in vitro cyclopropanation reactions, P450_(BM3) variants were used in purified form. Enzyme batches were prepared as follows. One liter TB_(amp) was inoculated with an overnight culture (100 mL, LB_(amp)) of recombinant E. coli BL21(DE3) cells harboring a pCWori plasmid encoding the P450 variant under the control of the tac promoter. After 3.5 h of incubation at 37° C. and 250 rpm shaking (OD₆₀₀ ca. 1.8), the incubation temperature was reduced to 25° C. (30 min), and the cultures were induced by adding IPTG to a final concentration of 0.5 mM. The cultures were allowed to continue for another 24 hours at this temperature. After harvesting the cells by centrifugation (4° C., 15 min, 3,000×g), the cell pellet was stored at −20° C. until further use but at least for 2 h. The cell pellet was resuspended in 25 mM Tris.HCl buffer (pH 7.5 at 25° C.) and cells were lysed by sonication (2×1 min, output control 5, 50% duty cycle; Sonicator, Heat Systems—Ultrasonic, Inc.). Cell debris was removed by centrifugation for 20 min at 4° C. and 27,000×g and the supernatant was subjected to anion exchange chromatography on a Q Sepharose column (HiTrap™ Q HP, GE Healthcare, Piscataway, N.J.) using an AKTAxpress purifier FPLC system (GE healthcare). The P450 (or P411) was eluted from the Q column by running a gradient from 0 to 0.5 M NaCl over 10 column volumes. The P450 (or P411) fractions were collected and concentrated using a 30 kDa molecular weight cutoff centrifugal filter and buffer-exchanged with 0.1 M phosphate buffer (pH

=8.0). The purified protein was flash-frozen on dry ice and stored at −20° C.

For crystallization experiments, a two-step purification was performed using the AKTAxpress purifier FPLC system. Frozen cell pellets containing expressed, 6×His tagged heme domains were resuspended in Ni-NTA buffer A (25 mM Tris.HCl, 200 mM NaCl, 25 mM imidazole, pH 8.0, 0.5 mL/gcw) and lysed by sonication (2×1 min, output control 5, 50% duty cycle). The lysate was centrifuged at 27,000×g for 20 min at 4° C. to remove cell debris. The collected supernatant was first subjected to a Ni-NTA chromatography step using a Ni sepharose column (H isTrap-HP, GE healthcare, Piscataway, N.J.). The P450 (or P411) was eluted from the Ni sepharose column using 25 mM Tris.HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Ni-purified protein was buffer exchanged into 25 mM Tris.HCl pH 7.5 using a 30 kDa molecular weight cutoff centrifugal filter and subsequently loaded onto a Q sepharose column (HiTrap™ Q HP, GE healthcare, Piscataway, N.J.) and purified to homogeneity by anion exchange. The P450 (or P411) was eluted from the Q column by running a gradient from 0 to 0.5 M NaCl over 10 column volumes. P450 (or P411) fractions were collected and buffer exchanged into 25 mM Tris.HCl pH 7.5, 25 mM NaCl. The purified protein was concentrated with a 30 kDa molecular weight cut-off centrifugal filter to approximately 10 mg mL⁻¹. Aliquots (50 μL) were flash frozen on dry ice and stored at −80° C. until needed.

Mutations in cyclopropanation catalysts are reported with respect to wild-type P450_(BM3). The heme domain comprises the first 462 amino acids in the P450_(BM3) sequence. P411_(BM3)=P450_(BM3)+C400S. P450_(BM3)-CIS=P450_(BM3)+V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A A290V, L353V, I366V, E442K. P411_(BM3)-CIS=P450_(BM3)-CIS+C400S.

Protein Crystallography.

P450_(BM3-heme)-CIS and P411_(BM3-heme)-CIS were crystallized by vapor diffusion. A 1:1 mixture of protein stock (10 mg/mL in 25 mM Tris.HCl pH 7.5, 25 mM NaCl) and mother liquor was combined in 24 well sitting drop plates (Hampton Research). Optimal crystallization conditions for P450_(BM3-heme)-CIS were found in 0.1 M sodium cacadolyte, pH 5.7, 0.14 MgCl₂ and 17% PEG 3350. P450_(BM3-heme)-CIS crystals typically grew over a span of 7-14 days. P411_(BM3-heme)-CIS crystals optimally formed in 0.1 M Bis-Tris, pH 5.3, 0.2 M sodium formate and 18% PEG 3350. Initial P411_(BM3-heme)-CIS drops are marked with a dense layer of protein precipitate; however, after 36-48 hours, noticeable protein crystals were observed underneath the precipitate layer.

X-Ray Data Collection and Protein Structure Determination.

X-ray diffraction data were collected at the General Medical Sciences and Cancer Institutes Structural Biology Facility (GM/CA) at the Advanced Photon Source (APS, Argonne National Laboratory) using beamline ID23-D and a MAR300CCD detector. Data were collected at 100K and a wavelength of 1.033 Å. Data collection statistics are listed in Table 38. Diffraction datasets were integrated with XDS (Kabsch, W. Acta Crystallogr., Sect. D: Biol. Crystallogr. D 66, 133-144 (2010)) and scaled using SCALA (Evans, P. Acta Crystallogr., Sect. D: Biol. Crystallogr. D 62, 72-82 (2006)). Initial phases were determined by molecular replacement against the closed form of wild type P450_(BM3-heme) structure taken from PDB 1JPZ (Haines, D. C. et al. Biochemistry 40, 13456-13465 (2001)), chain B using MOLREP software (Vagin, A. & Teplyakov, A. Journal of Applied Crystallography 30, 1022-1025 (1997)), a component of the CCP4 crystallography software suite (Bailey, S. Acta Crystallogr., Sect. D: Biol. Crystallogr. D 50, 760-763 (1994)). Refinement was accomplished by iterative cycles of manual model building within COOT (Emsley, P. & Cowtan, K. Acta Crystallogr., Sect. D: Biol. Crystallogr. D 60, 2126-2132 (2004)) and automated refinement using REFMAC (Murshudov, G. N. et al. Acta Crystallogr., Sect. D: Biol. Crystallogr. D 53, 240-255 (1997)) within CCP4. Final cycles of REFMAC refinement included TLS parameters. Non-crystallographic symmetry constraints were not used during refinement. Model quality was assessed using the ‘complete validation’ tool inside of the PHENIX software suite (Adams, P. D. et al. Acta Crystallogr., Sect. D: Biol. Crystallogr. D 66, 213-221 (2010)). Simulated annealing omit maps were also calculated using Phenix. Ramachandran outliers generally lie in poorly structured loops connecting P450_(BM3-heme) F and G helices. These residues are often missing or marked by poor density in these and other P450_(BM3-heme) structures within the protein database. All protein structure figures and alignments were generated using PyMol software (The PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC.).

Potentiometric Titrations.

Enzyme samples were buffer-exchanged into 100 mM KPO₄, 100 mM KCl, pH 7.4, and deoxygenated via 4×20 gentle pump-backfill cycles with argon, with care taken to avoid bubbling. Potentiometric redox titrations were performed in an anaerobic glove box, using a quartz spectroelectrochemical cell with path length of 1 mm, platinum mesh working electrode, platinum wire counter electrode, and a Ag/AgCl electrode (Bioanalytical Systems, Inc.) was used as the reference (Ag/AgCl vs NHE: +197 mV). Protein solutions consisted of approximately 600 μl of 50-100 μM protein with the following mediators added to ensure electrochemical communication between the protein and electrode: methyl viologen (5 μM), benzyl viologen (10 μM) and 2-hydroxy-1,4-napthaquinone (20 μM). Enzyme samples were titrated using sodium dithionite (reduction) and potassium ferricyanide (re-oxidation). The open circuit potential of the cell was monitored (WaveNow potentiostat, Pine Research Instrumentation) over a 10 minute equilibration period, and spectra were recorded using a Ocean Optics spectrometer (USB2000+). The reduction potentials (E^(o)′) were determined by fitting the data to the one-electron Nernst equation.

Typical Procedure for In Vitro Small-Scale Cyclopropanation Bioconversions Under Anaerobic Conditions.

Small-scale reactions (400 μL) were conducted in 2 mL crimp vials (Agilent Technologies, San Diego, Calif.). P450 solution (80 μL, 100 μM) was added to the vial with a small stir bar before crimp sealing with a silicone septum. Phosphate buffer (260 μL, 0.1 M, pH=8.0) and 40 μL of a solution of the reductant (100 mM Na₂S₂O₄, or 20 mM NADPH) were combined in a larger crimp-sealed vial and degassed by bubbling argon through the solution for at least 5 mM. In the meantime, the headspace of the 2 mL reaction vial with the P450 (or P411) solution was made anaerobic by flushing argon over the protein solution (with no bubbling). When multiple reactions were conducted in parallel, up to 8 reaction vials were degassed in series via cannulae. The buffer/reductant solution (300 μL) was syringed into the reaction vial, while under argon. The gas lines were disconnected from the reaction vial before placing the vials on a plate stirrer. A 40× styrene solution in MeOH (10 μL, typically 1.2 M) was added to the reaction vial via a glass syringe, and left to stir for about 30 s. A 40×EDA solution in MeOH was then added (10 μL, typically 400 mM) and the reaction was left stirring for the appropriate time. The final concentrations of the reagents were typically: 30 mM styrene, 10 mM EDA, 10 mM Na₂S₂O₄, 20 μM P450. The reaction was quenched by adding 30 μL HCl (3M) via syringe to the sealed reaction vial. The vials were opened and 20 μL internal standard (20 mM 2-phenylethanol in MeOH) was added followed by 1 mL ethyl acetate. This mixture was transferred to a 1.8 mL Eppendorf tube which was vortexed and centrifuged (16,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by chiral phase GC.

Determination of Initial Rates.

A slightly modified work-up was implemented for kinetic experiments. The reactions were quenched after the set time by syringing 1 mL EtOAc to the closed vials and immediately vortexing the mixture. The vials were then opened and 20 μL internal standard was added. The mixture was transferred to a 1.8 mL Eppendorf tube, vortexed and centrifuged (16,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed by GC. Both styrene and EDA concentrations were varied in the presence of the enzymes expressed as the heme-domain (0.5 or 1.0 μM BM3-CIS_(heme)). Reactions were set up in phosphate buffer (pH=8.0) with Na₂S₂O₄ as the reductant at 298 K, and were worked-up as described herein. Three time points were taken and used to determine the rate of product formation by GC (cyclosil-B 30 m×0.32 mm×0.25 μm): oven temperature=100° C. 5 min, 5° C./min to 200° C., 20° C./min to 250° C., 250° C. for 5 min. Elution time: cis-cyclopropanes (19.20 min and 19.33 min), trans-cyclopropanes (20.44 min). Apparent kinetic constants were determined by fitting the data to the standard Michaelis-Menten model.

Media and Cell Cultures for In Vivo Cyclopropanation.

E. coli [BL21(DE3)] cells were grown from glycerol stock overnight (37° C., 250 rpm) in 5 ml M9Y medium [1 L of 5×M9 medium contains 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 5.0 g NH₄C1, 0.24 g MgSO₄, and 0.01 g CaCl₂. 1 L M9Y contains 200 mL 5×M9, 800 mL deionized water, 15 g yeast extract, 1 mL micronutrients, and 0.1 mg mU¹ ampicillin). The pre-culture was used to inoculate 45 mL of M9Y medium in a 125 mL Erlenmeyer flask and this culture was incubated at 37° C., 250 rpm for 2 h and 30 min. At OD₆₀₀=1.2, the cultures were cooled to 25° C. and the shaking was reduced to 160 rpm before inducing with IPTG (0.25 mM) and δ-aminolevulinic acid (0.25 mM). Cultures were harvested after 20 h and resuspended (OD₆₀₀=30) in nitrogen-free M9 medium (1 L: 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 0.24 g MgSO₄, 0.01 g CaCl₂, 1 mL micronutrients). The micronutrient solution contains 0.15 mM (NH₄)₆Mo₇O₂₄, 20.0 mM H₃BO₃, 1.5 mM CoCl₂, 0.5 mM CuSO₄, 4.0 mM MnCl₂, and 0.5 mM ZnSO₄. Aliquots of the cell suspension were used for determination of the cell dry weight (cdw, 2 mL) and P450 (or P411) expression level (3 mL).

Small-Scale Whole-Cell Bioconversions.

Reaction conditions were as follows: 2 eq styrene, 1 eq EDA, 0.2 eq glucose, E. coli whole-cells in aqueous nitrogen-free M9 minimal medium and 5% MeOH cosolvent under anaerobic conditions for twelve hours at 298 K. E. coli cells (OD₆₀₀=30, 425 μL) were made anaerobic by bubbling argon through the cell suspension in a crimped 2 mL vial. A degassed solution of glucose (50 μL, 20 mM) was added to the cells before adding EDA (12.5 μL of a 400 mM solution in MeOH) and olefin (12.5 μL of a 800 mM solution in MeOH). The reactions were stirred at room temperature for the appropriate and were worked up by adding 20 μL of the internal standard (20 mM 2-phenylethanol) and extracting with 1 mL ethyl acetate. The organic layer was dried over Na₂SO₄ before analyzing the product mixture by chiral phase GC. Yields, diastereomeric ratios, and enantiomeric excess were determined by GC analysis. Yields based on EDA.

Preparative-Scale Whole-Cell Bioconversions.

E. coli [BL21(DE3)] cells were grown from glycerol stock overnight (37° C., 250 rpm) in 50 ml M9Y medium. The pre-culture was used to inoculate two 475 mL of M9Y medium in two 1 L Erlenmeyer flask (using 25 mL each) and this culture was incubated at 37° C., 250 rpm for 2 h and 30 min. At (OD₆₀₀=1.8, the cultures were cooled to 25° C. and the shaking was reduced to 150 rpm before inducing with IPTG (0.25 mM) and 6-aminolevulinic acid (0.25 mM). Cultures were harvested after 24 h and resuspended (OD₆₀₀=75) in nitrogen-free M9 medium. E. coli cells (OD₆₀₀=70, 53.6 mL) were made anaerobic by bubbling argon through the cell suspension in a 500 mL sealed round bottom flask. A degassed solution of glucose (1.4 mL, 500 mM) was added to the cells before adding EDA (1.36 mL, 85% EDA in DCM as packaged by Sigma Aldrich) and styrene (2.5 mL, neat). The reaction was stirred at room temperature under positive argon pressure for 24 h. The crude mixture was poured into three 50 mL conical tubes and the reaction was quenched by the addition of HCl (1 mL, 3 M) to each tube. The aqueous mixtures were extracted with 1:1 EtOAc:hexanes (20 mL each) and centrifuged (5000 rpm, 5 min). The organics were collected and this extraction sequence was performed two more times. The organics were combined, dried over Na₂SO₄ then concentrated. Excess styrene was removed via azeotrope with H₂O/benzene and 1.85 g of crude product was isolated. Cis/trans selectivity of the reaction was determined via gas chromatography of this crude mixture. Column chromatography of the crude product with 8% Et₂O/hexanes afforded the desired products as a mixture of cis and trans isomers (1.63 g combined, 78% yield). Based on comparison of crude and purified yields, the crude product was approximately 88% pure. NMR of the isolated products were identical to those reported in the literature (Coelho, P. S. et al. Science 339, 307-310 (2013)).

Time Course of In Vivo and In Vitro Reactions.

Following the procedure for small scale bioconversions, a series of in vivo and in vitro reactions were set up and EDA was added to each sample at time 0 hours. Time points were taken at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 hours. Each reported yield reflects an average of two independent reactions that were allowed to stir for the indicated amount of time. The error bars shown reflect the two unaveraged data points. Yields of each reaction were determined by GC.

Thermostability Measurements.

Duplicate measurements were taken for all values reported in FIG. 30. Purified P450 (or P411) solutions (4 μM, 200 μL) were heated in a thermocycler (Eppendorf) over a range of temperatures (40-70° C.) for 10 min followed by rapid cooling to 4° C. for 1 min. The precipitate was removed by centrifugation. The concentration of folded P450 (or P411) remaining in the supernatant was measured by CO-difference spectroscopy. The temperature at which half of the protein was denatured (T₅₀) was determined by fitting the data to the equation:

${f(T)} = {\frac{100}{1 + ^{- {a{({\frac{1}{T} - \frac{1}{T_{50}}})}}}}.}$

Accession Codes:

Atomic coordinates and structure factors have been deposited with the PDB (accession codes: 4H23 and 4H24).

Example 5 Enzymatic Synthesis of Milnacipran and Levomilnacipran

Variants of cytochrome P450_(BM3) (CYP102A1 or BM3) and whole cells expressing these enzymes have previously been used for the cyclopropanation of alphα-substituted styrenes with ethyl diazoacetate (EDA) (Coelho, P. S. et al. Science 339:307-310 (2013); Coelho, P. S. et al., Nat. Chem. Bio., 9:485-487 (2013)). This example illustrates the application of this method to the concise and stereoselective synthesis of milnacipran [racemic Z-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamide] and levomilnacipran [(1S,2R)-milnacipran] via both intermolecular and intramolecular olefin cyclopropanation. This example also illustrates the use of the method described herein to prepare aryl and amide derivatives of levomilnacipran as well as other drugs with similar chemical structure, such as bicifadine and DOV-216,303.

BACKGROUND

Milnacipran is a drug used to treat major depressive disorder (MDD) and fibromyalgia (a long-lasting condition that may cause pain, muscle stiffness and tenderness, tiredness, and difficulty falling asleep or staying asleep). Milnacipran is a selective serotonin (5-HT) and norepinephrine (NE) reuptake inhibitor (SSNRI) that works by blocking the 5-HT and NE transporters, thus increasing the extracellular levels of these two monoamine neurotransmitters in the brain. Levomilnacipran, the most psychoactive stereoisomer of milnacipran, is currently in phase III clinical trials for the treatment of major depressive disorders.

Several strategies have been developed for the synthesis of milnacipran (Pineiro-Nunez, M. in The Art of Drug Synthesis (ed J. J. Li D. S. Johnson) Ch. 14, 205-207 (Wiley, 2007)). Because the commercial drug is a racemate, the synthetic routes are simply concerned with the relative stereochemistry around the cyclopropane ring. The original synthesis of milnacipran employed an alkylation and epoxide ring-opening sequence starting from 2-phenylacetonitrile and 2-(chloromethyl)oxirane (FIG. 36A) (Bonnaud, B. et al., European Patent No. 0200638B1; Bonnaud, B. et al., J. Medicinal Chemistry 30, 318-325 (1987)), which yielded a mixture of Z and E cyclopropanes. Lactonization of the resulting products under thermal conditions yielded key intermediate 1 ((1S,5R)-1-phenyl-3-oxabicyclo[3.1.0]hexan-2-one), which contained the requisite Z-geometry at stereocenters 1 and 2. This intermediate has been converted to milnacipran using three routes outlined in FIG. 36B. In Route A, phthalimide ring opening yielded 2, which was readily converted to milnacipran after amidation and pthalimide deprotection. In Route B, 1 was opened with hydrobromic acid and treated with thionyl chloride to generate doubly functionalized intermediate 3 (Mouzin, G. et al., Synthesis, 4, 304-305 (1978)). Stepwise treatment with diethylamine and potassium phtalimide generated an intermediate, which was common to the synthesis in Route A. In Route C, direct amide formation using n-BuLi and HNEt₂ yielded 4 which could be converted to milnacipran after azide nucleophilic substitution followed by hydrogenation (Shuto, S. et al., J. Med. Chem. 38, 2964-2968 (1995)). FIG. 36C shows an entirely different approach to milnacipran synthesis. The general strategy is based on position selective deprotonation of the commercially available cyclopropane carboxamide 5 (Zhang, M.-X. et al., Angew. Chem. Int. Ed., 41, 2169-2170 (2002)).

Enantioselective syntheses of levomilnacipran have focused on asymmetric methods for formation of the key intermediate 1. FIG. 37A shows the first route which employed a chiral dirhodium(II) tetrakis[methyl 2-oxaazetidine-4(S)-carboxylate], Rh2(4S-MEAZ)₄ complex for intramolecular cyclopropanation of 6 and produced 1 in 68% enantioselectivity (Doyle, M. P. et al., Org. Lett. 2, 1145 (2000)). More recently, Alliot et al. prepared 1 from 2-phenylacetic acid using asymmetric alkylation using a chiral tetramine auxiliary (Alliot, J. et al., Chem. Commun. 48, 8111-8113 (2012)) (FIG. 37B). Treatment of the alkylation product with I₂ produced a masked halohydrin 7, which underwent esterification and epoxide ring formation under basic conditions. The overall sequence produced 1 in 41% yield and 88% ee.

Results

In certain aspects, this example describes the formal synthesis of levomilnacipran via direct enantioselective cyclopropanation of N,N-diethyl-2-phenylacrylamide by diazoacetonitrile or ethyl diazoacetate (EDA). Selective nitrile hydrogenation or ester to alcohol reduction then provides levomilnacipran or product 8 (FIG. 38), which is converted to levomilnacipran following previously published amination protocols (Alliot, J. et al., Chem. Commun. 48, 8111-8113 (2012)). Alternatively, an intramolecular cyclopropanation route such as the one shown in FIG. 37A is employed for synthesis of intermediate 1 using P450 enzymes. In both strategies, all stereocenters in the final product would be constructed via enzymatic cyclopropanation.

These methods are an improvement over previous racemic synthesis because the proposed routes advantageously provide the more psychoactive isomer of milnacipran. Furthermore, the intermolecular route proposed in FIG. 38 is the first route to employ late stage cyclopropanation for synthesis of milnacipran and is highly convergent as the amide moiety is installed prior to cyclopropanation. The intramolecular route is advantageous over previously reported routes because it is concise and enables the use of P450_(BM3) or whole cells expressing these proteins in place of costly rhodium complexes with highly designed synthetic ligands.

The preceding examples show that P450 are excellent catalysts for carbene transfer from diazo compounds to olefins to form cyclopropanes. This reaction has been explored for a variety of substituted olefins and in particular, the present examples show that cyclopropanation can be performed on mono- and disubstituted styrenes. Additionally, functional groups and substitution on the aryl ring of styrene is also well-tolerated by this reaction including electron donating groups such as methoxy and electron withdrawing groups such as trifluoromethyl. This example demonstrates that 1) amide and ester functionalities are compatible with P450-mediated cyclopropanation and 2) significant steric bulk can be tolerated in the alpha position of styrene.

This example shows that P450s are competent catalysts for the cyclopropanation of substituted styrenes of the general form 9, where R=ester or amide. Treatment of the acrylate ester 9a (20 mM) and EDA (8.5 mM) with WT-T268A-C400S (2 mutations from wild-type BM3, herein called P411-T268A, 10 μM, where “P411” denotes the C400S mutation) and sodium dithionite (10 mM) yielded appreciable amounts of the corresponding product 10a. BM3-CIS-C400S (14 mutations from wild-type, herein called P411-CIS) was also able to catalyze the cyclopropanation of the acrylamide 9b, albeit at very low conversions (<1%). A screen of axial mutants at position C400 in BM3-CIS yielded variant BM3-CIS-AxH (where “AxH” denotes a Cys to His substitution in the 400 position of BM3), which exhibited high activity in the reaction of 9b and EDA to provide 10b at 91% yield, 93:7 Z:E diastereoselectivity, and 42% enantioselectivity after 16 h at 25° C. (Table 48).

TABLE 48 P450-screen for cyclopropanation of milnacipran precursors.

Entry Catalyst Substrate Yield (%) E/Z Ratio 1 BM3-CIS 9a <5 N/A 2 P411-T268A 9a >80 43:57 3 P411-T268A 9b 1 N/A 4 BM3-CIS 9b 0 N/A 5 P411-CIS 9b 0 N/A 6 BM3-CIS-AxD 9b 44 13:87 7 BM3-CIS-AxY 9b 38 16:84 8 BM3-CIS-AxK 9b 45 17:83 9 BM3-CIS-AxH 9b 91  7:93 10 BM3-CIS-AxM 9b 46 17:83 11 Hemin 9b 3 18:82 “P411” denotes the C400S mutation and “Ax” denotes a mutation to all other amino acid at the axial position (position 400 in BM3). Reactions were carried out under anaerobic conditions with 8.5 mM EDA, 20 mM 9a or 10 mM 9b, 10 μM P450, and 10 mM Na₂S₂O₄.

Intact whole cells expressing BM3-CIS-AxH can also be used for the cyclopropanation of 9b. E. coli cells expressing P450 were grown in Hyperbroth then resuspended in nitrogen-free M9 minimal media at pH 7 to OD₆₀₀=30 (7.8 g_(CDW)/L). Reaction of 9b and EDA in the presence of these cells and glucose under anaerobic conditions provided 10b in 82% yield. Optimization of BM3-CIS-AxD by site saturation mutagenesis at position T438 yielded BM3-CIS-AxH-T438W, which catalyzed the reaction to 52% yield and 78% enantioselectivity in vivo.

The preceding examples demonstrate that aryl substitution and alpha substitution on styrene are well tolerated by P450 cyclopropanation. Thus, the method described herein may also be used to prepare aryl and amide derivatives of levomilnacipran as well as other drugs with similar chemical structure, such as bicifadine and DOV-216,303 (FIG. 40). For instance, bicifadine and DOV-216,303 can both be prepared using alphα-styrenyl amides and esters similar to the substrates shown herein. Again, cyclopropanation constructs both stereocenters and subsequent functional group manipulation and cyclization would yield bicifadine or DOV-216,303.

Materials and Methods

Procedure for Reactions with Isolated Enzymes:

Small-scale reactions (400 μL) were conducted in 2 mL crimp vials (Agilent Technologies, San Diego, Calif.). P450 solution (60 μL, 67 μM) was added to the vial before crimp sealing with a silicone septum. A solution of Na₂S₂O₄ (12.5 mM) in phosphate buffer (0.1 M, pH=8.0) was prepared and sealed in a larger crimp-sealed vial and degassed by bubbling argon through the solution for 5 min. In the meantime, the headspace of the 2 mL reaction vial with the protein solution was made anaerobic by flushing argon over the vial headspace (with no bubbling). When multiple reactions were conducted in parallel, up to 8 reaction vials were degassed in series via cannulae. The buffer/reductant solution (320 μL) was syringed into the reaction vial, while under argon. A 40× styrene solution in EtOH (10 μL, 400 mM) was added to the reaction vial via a glass syringe followed by a 40×EDA solution in EtOH (10 μL, 400 mM). The reactions were shaken on a shake plate at 350 rpm for 16-20 h. The final concentrations of the reagents were typically: 10 mM styrene, 8.5 mM EDA, 10 mM Na₂S₂O₄, 10 μM protein. The reaction was quenched by adding 30 μL HCl (3M) via syringe to the sealed reaction vial. The vials were opened and 20 μL internal standard (20 mM 2-phenylethanol in EtOH) was added followed by 1 mL cyclohexane. This mixture was transferred to an eppendorf tube which was vortexed and centrifuged (13,000×g, 1 min). The top organic layer was dried over an anhydrous sodium sulfate plug and analyzed for yield and enantioselectivity.

Procedure for Whole Cell Reactions:

E. coli cells (OD₆₀₀=30, 425 μL) were made anaerobic by bubbling argon through the cell suspension in a crimped 2 mL vial. A degassed solution of glucose (50 μL, 200 mM) was added to the cells before adding EDA (12.5 μL of a 400 mM solution in EtOH) and olefin (12.5 μL of a 400 mM solution in EtOH). The final concentrations of the reagents were typically: 10 mM styrene, 8.5 mM EDA, and 20 mM glucose. The reactions were shaken at room temperature for 16-20 h and were worked up by adding 20 μL of the internal standard (20 mM 2-phenylethanol) and extracting with 1 mL cyclohexane. The organic layer was dried over Na₂SO₄ then analyzed for yield and enantioselectivity.

Determination of Yield, Enantioselectivity and Absolute Chirality:

Yield for purified enzyme and whole cell reactions were determined via gas chromatography using a flame ionization detector, relative to phenethyl alcohol as internal standard (FIG. 39). Gas chromatography was performed using Agilent cycloSil-B column, 30 m×0.25 μm×0.32 mm, method: 90° C. (hold 2 min), 90-110 (6° C./min), 110-190 (40° C./min), and 190-280 (20° C./min): internal standard at 4.8 min, starting material at 10.4 min, and product at 14.8 min (E-isomer) and 15.4 min (Z-isomer). Independently synthesized product 10b was used to generate a GC calibration curve. Identity of the product was confirmed by proton NMR and relative stereochemistry of the major diastereomer was determined by Nuclear Overhauser effect spectroscopy (NOESY). Enantioselectivity was established by chiral HPLC (on ChiralPak AS column, eluting with 4% IPA/supercritical CO₂), using a racemic sample of 10b prepared from 9b, EDA, and hemin as reference. The absolute stereochemistry of 10b was established by polarimetry after reduction of 10b to 8 using LiBH₄. Comparison of the optical rotation of product to published specific rotation of 8 (Alliot, J. et al., Chem. Commun. 48, 8111-8113 (2012)) allowed assignment of stereocenters in position 1 and 2 (FIG. 38) as 1S, 2R, in agreement with levomilnacipran.

Amino Acid Sequences:

BM3-CIS contains the following mutations from wild type: V78A F87V P142S T175I A184V S226R H236Q E252G T268A A290V L353V I366V E442K. P411-CIS contains BM3-CIS and Cys to Ser at position 400 of BM3. BM3-CIS-A×X (where X=H, D, A, M, etc.) mutants contains Cys to X mutation at position 400 of BM3. The complete P450_(BM3) sequence is set forth in SEQ ID NO:1.

Example 6 Enzymatic Synthesis of Pyrethroid and Pyrethrin Insecticides

This example illustrates the synthesis of ethyl chrysanthemate using the cytochrome P450 catalysts of the present invention. The resulting ethyl chrysanthemate can be converted chemically or enzymatically to pyrethroid and pyrethrin insecticides.

Cytochrome P450 derived cyclopropanation catalysts show a measurable promiscuous activity towards the synthesis of pyrethric acids from ethyl diazoacetate (EDA) and diolefins. This example illustrates that P450 catalysts can make ethyl chrysanthemate from EDA and 2,5-dimethyl-2,4-hexadiene. Intact E. coli cells expressing a double mutant of wild-type cytochrome P450_(BM3)(CYP102A1), BM3-T268A-C400S, displayed measurable activity for the synthesis of ethyl chrysanthemate as shown in FIG. 41. Purified BM3-T268A-C400S was also capable of making chrysanthemate in vitro.

The reaction solution was quenched with acid (final concentration of 1% HCL) and extracted with ethyl acetate. 2-Phenylethanol was used an internal standard for analysis by gas-chromatography. An authentic sample of ethyl chrysanthemate was purchased from Sigmα-Aldrich. Enzymatic production of chrysanthemate was confirmed by GC-FID and GC-MS (FIGS. 42 and 43).

The resulting ethyl chrysanthemate can be converted chemically or enzymatically to pyrethroid and pyrethrin insecticides as shown in FIG. 44. For example, a lipase enzyme can be used to catalyze the transesterification reaction shown in FIG. 44( d).

Example 7 Cytochrome P450-Cam Catalyzed Cyclopropanation of Styrene

This example illustrates that cytochrome P450-Cam (Cyp101A1, SEQ ID NO:25) and its cis-axial C357S variant (P411-Cam, wherein the variant has a C357S substitution at the axial position in SEQ ID NO:25) are capable of catalyzing the cyclopranation of styrene using ethyl diazoacetate (EDA) as the carbene precursor, thereby resulting in ethyl 2-phenylcyclopropane-1-carboxylate.

E. coli BL21 (DE3) cells expressing cytochrome P450-Cam and P411-Cam from plasmid pET22 were grown from glycerol stock overnight (37° C., 250 rpm) in 5 ml Hyperbroth medium (54.0 g/L Hyperbroth mix as purchased from Athena Enzyme Systems, 0.1 μg/L ampicillin). The pre-culture was used to inoculate 45 mL of M9Y medium (per 1 L: 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 5.0 g NH₄C1, 0.24 g MgSO₄, 0.01 g CaCl₂, 1.5% yeast extract, 1 mL micronutrients (0.15 mM (NH₄)₆Mo₇O₂₄, 20.0 mM H₃BO₃, 1.5 mM CoCl₂, 0.5 mM CuSO₄, 4.0 mM MnCl₂, and 0.5 mM ZnSO₄), 0.1 mg mL⁻¹ ampicillin) in a 125 mL Erlenmeyer flask and this culture was incubated at 37° C., 250 rpm for 2 h and 30 min. At OD₆₀₀˜1.2, the cultures were cooled to 25° C. and the shaking was reduced to 160 rpm before inducing with IPTG (0.25 mM) and 6-aminolevulinic acid (0.25 mM). Cultures were harvested after 20 h and resuspended (OD₆₀₀=30) in nitrogen-free M9 medium (per 1 L: 31 g Na₂HPO₄, 15 g KH₂PO₄, 2.5 g NaCl, 0.24 g MgSO₄, 0.01 g CaCl₂, 1 mL micronutrients (0.15 mM (NH₄)₆Mo₇O₂₄, 20.0 mM H₃BO₃, 1.5 mM CoCl₂, 0.5 mM CuSO₄, 4.0 mM MnCl₂, and 0.5 mM ZnSO₄)). The micronutrient solution contains 0.15 mM (NH₄)₆Mo₇O₂₄, 20.0 mM H₃BO₃, 1.5 mM CoCl₂, 0.5 mM CuSO₄, 4.0 mM MnCl₂, and 0.5 mM ZnSO₄. Aliquots of the cell suspension were used for determination of the cell dry weight (2 mL) and P450 (or P411) expression level (4 mL).

Prior the styrene cyclopropanation using ethyl diazoacetate as the carbene precursor, the cells at specified cell density (see, Table 49) were made anaerobic by bubbling argon through the cell suspension in a crimped 2 mL vial. A degassed solution of glucose (50 μL, 20 mM) was added to the cells before adding EDA (12.5 μL of a 400 mM solution in MeOH) and styrene (12.5 μL, of a 1.2 M solution in MeOH). The reactions were shaken at room temperature for 16 h and were worked up by adding 204 of the internal standard (20 mM 2-phenylethanol) and extracting with 1 mL cyclohexane. The organic layer was analyzed by chiral phase GC.

Gas chromatography (GC) analyses were carried out using a Shimadzu GC-17A gas chromatograph, a FID detector, and an Agilent J&W cyclosil-B column (30 m×0.25 mm, 0.25 μm film) and 2-phenylethanol as an internal standard. Injector temperature=300° C., oven temperature=130° C. for 30 min, pressure=175 kPa. Elution time: cis-cyclopropanes [19.7 min (2R,1S) and 21.0 min (2S,1R)], trans-cyclopropanes [25.8 min (2R,1R) and 26.4 min (2S,1S)]. The ethyl 2-phenylcyclopropane-1-carboxylate product standard for the reaction of ethyl diazoacetate (EDA) with styrene was prepared as reported (A. Penoni et al., Eur. J. Inorg. Chem. 2003, 1452 (2003)). These standards and enzyme-prepared cyclopropanes demonstrated identical retention times in gas chromatograms when co-injected, confirming product identity. Absolute stereoconfiguration of cyclopropane enantiomers was determined by measuring optical rotation of purified cyclopropane products from preparative bioconversion reactions using enantioselective BM3 variants and referenced to values taken from reference (N. Watanabe et al., Heterocycles 42, 537 (1996)).

The cytochrome P450 concentration was determined from ferrous CO binding difference spectra of lysate using extinction coefficients of ε₄₅₀₋₄₉₀=91 mM⁻¹ cm⁻¹ for cysteine-ligated P450-Cam (T. Omura, R. Sato, J. Biol. Chem. 239, 2370 (1964)) and ε₄₁₁₋₄₉₀=103 mM⁻¹ cm⁻¹ for serine ligated P411-Cam (K. P. Vatsis et al., J. Inorg. Biochem. 91, 542 (2002)).

Results are shown in Table 49 and demonstrate that both P450-Cam and P411-Cam catalyze cyclopropropanation of styrene with EDA.

TABLE 49 P450-Cam and P411-Cam catalyzed cyclopropanation of styrene c_((styrene)) c_((EDA)) c_((P450 or P411)) c _((cells)) c_((product)) Yield Cis: ee_(cis) ee_(trans) Enzyme [mM] [mM] [μM] [g/L] [mM] [%] TTN Trans [%] [%] P450-Cam 20 8.5 9.6 5.4 4.8 56 480 82:18   43% 0 P411-Cam 20 8.5 1.0 8 2.4 28 2400 13:87  <5% <5%

Example 8 Cyclopropanation by Axial Mutants of P450-BM3

This example illustrates that cytochrome variants of P450-BM3 containing mutations at position 400, the axial heme coordination site, from Cys to Ala, Asp, His, Lys, Asn, Met, Thr, or Tyr, are capable of catalyzing the cyclopranation of styrene using ethyl diazoacetate (EDA) as the carbene precursor, resulting in ethyl 2-phenylcyclopropane-1-carboxylate. The convention “A×X”, wherein X is the single letter amino acid code of the amino acid at the axial position, can be used to describe this series of enzymes.

P450 were used as isolated and purified proteins. One liter TB_(amp) was inoculated with an overnight culture (25 mL, TB_(amp)) of recombinant E. coli BL21 cells harboring pET22 plasmid encoding the his6-tagged P450 variants under the control of the tac promoter. The cultures were shaken at 200 rpm at 37° C. for roughly 3.5 h or until an optical of density of 1.2-1.8 was reached. The temperature was reduced to 25° C. and the shake rate was reduced to 180 rpm for 20 min, then the cultures were induced by adding IPTG and aminolevulinic acid to a final concentration of 0.5 mM. The cultures were allowed to continue for another 20 hours at this temperature. Cells were harvested by centrifugation (4° C., 15 min, 3,000×g), and the cell pellet was stored at −20° C. for at least 2 h.

For the purification of his6-tagged P450s, the thawed cell pellet was resuspended in Ni-NTA buffer A (25 mM Tris.HCl, 200 mM NaCl, 25 mM imidazole, pH 8.0, 0.5 mL/gcw) and lysed by sonication (2×1 min, output control 5, 50% duty cycle). The lysate was centrifuged at 27,000×g for 20 min at 4° C. to remove cell debris. The collected supernatant was first subjected to a Ni-NTA chromatography step using a Ni Sepharose column (H isTrap-HP, GE healthcare, Piscataway, N.J.). The P450 was eluted from the Ni Sepharose column using 25 mM Tris.HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Ni-purified protein was buffer exchanged into 0.1 M phosphate buffer (pH=8.0) using a 30 kDa molecular weight cut-off centrifugal filter. Protein concentrations were determined by CO-assay as described above. For storage, proteins were portioned into 300 uL aliquots and stored at −80° C.

Reactions (400 μL) were conducted in 2 mL crimp vials (Agilent Technologies, San Diego, Calif.). P450 solution (80 μL, 100 μM) was added to the vial with a small stir bar before crimp sealing with a silicone septum. Phosphate buffer (260 μL, 0.1 M, pH=8.0) and 40 μL of a solution of the reductant (100 mM Na₂S₂O₄) were combined in a larger crimp-sealed vial and degassed by bubbling argon through the solution for at least 5 min. In the meantime, the headspace of the 2 mL reaction vial with the P450 solution was made anaerobic by flushing argon over the protein solution (with no bubbling). When multiple reactions were conducted in parallel, up to 8 reaction vials were degassed in series via cannulae. The buffer/reductant solution (300 μL) was syringed into the reaction vial, while under argon. The gas lines were disconnected from the reaction vial before placing the vials on a plate stirrer. A styrene solution in MeOH (10 pt, 800 mM) was added to the reaction vial via a glass syringe, and left to stir for about 30 s. A EDA solution in MeOH was then added (10 μL, 340 mM) and the reaction was left shaking for 16 h at room temperature. The final concentrations of the reagents were: 20 mM styrene, 8.5 mM EDA, 10 mM Na₂S₂O₄, 20 μM P450.

After 16 h, the vials were opened and 20 μL internal standard (20 mM 2-phenylethanol in MeOH) was added followed by 1 mL cyclohexane. This mixture was transferred to a 1.8 mL eppendorf tube which was vortexed and centrifuged (16,000×g, 1 min). The top organic layer was analyzed by chiral phase GC.

Gas chromatography (GC) analyses were carried out using a Shimadzu GC-17A gas chromatograph, a FID detector, and an Agilent J&W cyclosil-B column (30 m×0.25 mm, 0.25 μm film) and 2-phenylethanol as an internal standard. Injector temperature=300° C., oven temperature=130° C. for 30 min, pressure=175 kPa. Elution time: cis-cyclopropanes [19.7 min (2R,1S) and 21.0 min (2S,1R)], trans-cyclopropanes [25.8 min (2R,1R) and 26.4 min (2S,1S)]. The ethyl 2-phenylcyclopropane-1-carboxylate product standard for the reaction of ethyl diazoacetate (EDA) with styrene was prepared as reported (A. Penoni et al., Eur. J. Inorg. Chem. 2003, 1452 (2003)). These standards and enzyme-prepared cyclopropanes demonstrated identical retention times in gas chromatograms when co-injected, confirming product identity. Absolute stereoconfiguration of cyclopropane enantiomers was determined by measuring optical rotation of purified cyclopropane products from preparative bioconversion reactions using enantioselective BM3 variants and referenced to values taken from reference (N. Watanabe et al., Heterocycles 42, 537 (1996)).

The cytochrome P450 concentration was determined from hemechrome assay of purified protein using extinction coefficients of £₄₁₈=196 mM⁻¹ cm⁻¹ for the hemechrome complex (J. E. Falk, Porphyrins and Metalloporphyrins, Elsevier: Amsterdam, 1975. p 801-807).

Results are shown in Table 50 and demonstrate that axial mutants are active cyclopropanation catalysts.

TABLE 50 Cyclopropanation of styrene catalyzed by P450-BM3 axial mutants (5 uM) AA at c_((styrene)) c_((EDA)) c_((product)) Yield SEQ ID Enzyme name Backbone position 400 [mM] [mM] [mM] [%] TTN Cis:Trans NO: WT-AxA (heme) WT Ala 20 8.5 3.9 56 780 12:88 50 WT-AxD (heme) WT Asp 20 8.5 3.4 28 670 13:87 51 WT-AxH (heme) WT His 20 8.5 3.9 46 780  8:92 52 WT-AxK (heme) WT Lys 20 8.5 3.8 45 760 13:87 53 WT-AxM (heme) WT Met 20 8.5 3.3 39 650 11:89 54 WT-AxN (heme) WT Asn 20 8.5 4.3 51 870 13:87 55 BM3-CIS-T438S-AxA BM3-CIS-T438S Ala 20 8.5 2.8 33 560 10:90 56 BM3-CIS-T438S-AxD BM3-CIS-T438S Asp 20 8.5 2.2 26 430 16:84 57 BM3-CIS-T438S-AxM BM3-CIS-T438S Met 20 8.5 5.4 63 1070 69:31 58 BM3-CIS-T438S-AxY BM3-CIS-T438S Tyr 20 8.5 2.0 23 390 21:79 59 BM3-CIS-T438S-AxT BM3-CIS-T438S Thr 20 8.5 3.0 35 590 15:85 60 The term “(heme)” refers to a truncated variant of wild-type (WT) P450-BM3 comprising amino acids 1-463 of SEQ ID NO: 1 and the indicated point mutation at amino acid position 400, the axial heme coordination site. The term “BM3-CIS-T438S” refers to a full-length variant of WT P450-BM3 comprising the following amino acid substitutions in SEQ ID NO: 1: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, T438S, and E442K.

Example 9 Identification of Cys Axial Ligand in Cytochromes P450

This example illustrates how to identify the conserved cysteine residue in cytochrome P450 enzymes that serves as the heme axial ligand via sequence alignment.

The known cytochrome P450-BM3 axial ligand was used to identify the axial ligand in a new P450, CYP2D7 from Homo sapiens, GenBank accession number AA049806.1. To identify the axial ligand in this enzyme, a protein alignment algorithm provided by the National Institute of Health's NCBI blastp suite, version 2.2.28+(http://blast.ncbi.nlm.nih.gov/Blast.cgi; S. F. Altschul, et al. (1997), Nucleic Acids Res. 25:3389-3402; S. F. Altschul, et al. (2005) FEBS J. 272:5101-5109) was used using the following parameters: E value=10, word size=3, Matrix=Blosum62, and Gap opening=11 and gap extension=1, and conditional compositional score matrix adjustment. Upon entering the protein sequences for P450-BM3 (SEQ ID NO:1) as subject and CYP2D7 as query and requesting an alignment between the two sequences, blastp returned a proposed alignment that included the BM3 C400 site (FIG. 45A). On the last row of this alignment, a semi-conserved region including a cysteine was apparent. The cysteine on the lower subject line is BM3's C400 axial ligand. Accordingly, the C461 above in the query line can be identified as the axial ligand in the CYP2D7 protein.

As a second example, the known P450-BM3 axial ligand was used to identify the axial ligand in P450C27, a mitochondrial P450 from Rattus norvegicus, GenBank accession number AAB02287.1. Upon entering the protein sequences for P450-BM3 (SEQ ID NO:1) as subject and P450C27 as query into blastp and requesting an alignment between the two sequences using the parameters described above, a proposed alignment that included the BM3 C400 site (FIG. 45B) was returned.

On the second to last row of this alignment, a semi-conserved region including a cysteine was apparent. The cysteine on the lower subject line is BM3's C400 axial ligand. Accordingly, the C478 above in the query line can be identified as the axial ligand in the P450C27 protein.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. 

What is claimed is:
 1. A reaction mixture for producing a cyclopropanation product, the reaction mixture comprising an olefinic substrate, a carbene precursor, and a heme enzyme.
 2. The reaction mixture of claim 1, wherein the cyclopropanation product is a compound according to Formula I:

wherein: R¹ is independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(1a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃; R² is independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(2a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃; wherein R^(1a) and R^(2a) are independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl and -L-R^(c), wherein each L is selected from the group consisting of a bond, —C(R^(L))₂—, and —NR^(L)—C(R^(L))₂—, each R^(L) is independently selected from the group consisting of H, C₁₋₆ alkyl, halo, —CN, and —SO₂, and each R^(C) is selected from the group consisting of optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl; and R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H, C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted C₁-C₆ alkoxy, halo, hydroxy, cyano, C(O)N(R⁷)₂, NR⁷C(O)R⁸, C(O)R⁸, C(O)OR⁸, and N(R⁹)₂, wherein each R⁷ and R⁸ is independently selected from the group consisting of H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl; and each R⁹ is independently selected from the group consisting of H, optionally substituted C₆₋₁₀ aryl, and optionally substituted 6- to 10-membered heteroaryl, or two R⁹ moieties, together with the nitrogen atom to which they are attached, can form 6- to 18-membered heterocyclyl; or R³ forms an optionally substituted 3- to 18-membered ring with R⁴; or R⁵ forms an optionally substituted 3- to 18-membered ring with R⁶; or R³ or R⁴ forms a double bond with R⁵ or R⁶; or R³ or R⁴ forms an optionally substituted 5- to 6-membered ring with R⁵ or R⁶.
 3. The reaction mixture of claim 2, wherein: R¹ is C(O)O-LR^(c); R² is selected from the group consisting of H and optionally substituted C₆₋₁₀ aryl; and R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H, optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, optionally substituted C₆₋₁₀ aryl and halo, or R³ forms an optionally substituted 3- to 18-membered ring with R⁴; or R⁵ forms an optionally substituted 3- to 18-membered ring with R⁶.
 4. The reaction mixture of claim 2, wherein the cyclopropanation product is a compound according to Formula II:

wherein R^(1a) is C₁₋₆ alkyl and R² is selected from the group consisting of H and optionally substituted C₆₋₁₀ aryl.
 5. The reaction mixture of claim 4, wherein the cyclopropanation product is selected from the group consisting of:

wherein: X¹ is selected from the group consisting of H, optionally substituted C₁₋₆ alkyl, haloC₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, optionally substituted C₁₋₆ alkylthio, C₁₋₆ alkylsilyl, halo, and cyano; X² is selected from the group consisting of H, chloro, and methyl; X³ is selected from the group consisting of H, methyl, halo, and CN; each X⁴ is independently halo; each X⁵ is independently selected from the group consisting of methyl and halo, X⁶ is selected from the group consisting of halo, optionally substituted C₁₋₆ alkyl, and optionally substituted C₁₋₆ alkoxy; X⁷ is selected from the group consisting of H, methyl, and halo; X⁸ is selected from the group consisting of H, halo, and optionally substituted C₁₋₆ alkyl; X⁹ is selected from the group consisting of H, halo, optionally substituted C₁₋₆ alkyl, C(O)O—(C₁₋₆ alkyl), C(O)—N(C₁₋₆ alkyl)₂, and cyano; and Z¹, Z², and Z³ are independently selected from the group consisting of H, halo, optionally substituted C₁₋₆ alkyl, and optionally substituted C₆₋₁₀ aryl; or Z¹ and Z² are taken together to form an optionally substituted 5- to 6-membered cycloalkyl or heterocyclyl group.
 6. The reaction mixture of claim 4, wherein the cyclopropanation product is an intermediate of a compound according to Formula III:

wherein: L is selected from the group consisting of a bond, —C(R^(L))₂—, and —NR^(L)—C(R^(L))₂—, each R^(L) is independently selected from the group consisting of H, —CN, and —SO₂, and R^(1c) is selected from the group consisting of optionally subsistuted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl.
 7. The reaction mixture of claim 6, wherein the moiety L-R^(1c) has a structure selected from the group consisting of:

wherein: each Y¹ is independently selected from the group consisting of optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, phenyl, and (phenyl)C₁₋₆ alkoxy; each Y² is independently selected from the group consisting of halo, optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₁₋₆ alkoxy, and nitro; each Y³ is independently optionally substituted C₁₋₆ alkyl; each Y⁴ is independently selected from the group consisting of optionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, C₆₋₁₀ aryl-C₁₋₆ alkyl, furfuryl, C₁₋₆ alkoxy, (C₂₋₆ alkenyl)oxy, C₁₋₁₂ acyl, and halo; Y⁵ is selected from the group consisting of optionally substituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, and halo, the subscript m is an integer from 1 to 3, the subscript n is an integer from 1 to 5, the subscript p is an integer from 1 to 4, the subscript q is an integer from 0 to 3, and the wavy line at the left of the structure represents the point of connection between the moiety -L-R¹⁶ and the rest of the compound according to Formula III.
 8. The reaction mixture of claim 1, wherein the cyclopropanation product is a compound having a structure according to the formula:

wherein: R^(1a) is optionally substituted C₁₋₆ alkyl, and R⁵ and R⁶ are independently selected from the group consisting of H, optionally substituted C₁₋₆ alkyl, optionally substituted C₆₋₁₀ aryl, C(O)N(R⁷)₂, C(O)OR⁸ and NR⁷C(O)R⁸, and wherein each R⁷ and R⁸ is independently selected from the group consisting of H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl.
 9. The reaction mixture of claim 8, wherein the cyclopropanation product has a structure selected from the group consisting of:


10. The reaction mixture of claim 1, wherein the cyclopropanation product is an intermediate of milnacipran, levomilnacipran, cilastain, or sitafloxacin.
 11. The reaction mixture of claim 10, wherein the intermediate of cilastain has a structure according to the formula:

wherein: R is OR^(1a) or N(R⁷)₂, wherein R^(1a) is selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl and -L-R^(C), wherein each L is selected from the group consisting of a bond, —C(R^(L))₂—, and —NR^(L)—C(R^(L))₂—, each R^(L) is independently selected from the group consisting of H, C₁₋₆ alkyl, halo, —CN, and —SO₂, and each R^(C) is selected from the group consisting of optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl; and R⁷ is selected from the group consisting of H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl.
 12. The reaction mixture of claim 1, wherein the olefinic substrate is selected from the group consisting of an alkene, a cycloalkene, and an arylalkene.
 13. The reaction mixture of claim 12, wherein the arylalkene is a styrene.
 14. The reaction mixture of claim 13, wherein the styrene has the formula:

wherein: R³ is selected from the group consisting of H, optionally substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, C(O)N(R⁷)₂, C(O)OR⁸, N(R⁹)₂, halo, hydroxy, and cyano; wherein each R⁷ and R⁸ is independently selected from the group consisting of H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl; and each R⁹ is independently selected from the group consisting of H, optionally substituted C₆₋₁₀ aryl, and optionally substituted 6- to 10-membered heteroaryl, or two R⁹ moieties, together with the nitrogen atom to which they are attached, can form 6- to 18-membered heterocyclyl; R⁵ and R⁶ are independently selected from the group consisting of H, optionally substituted C₁₋₆ alkyl, and halo; R¹⁰ is selected from the group consisting of optionally substituted C₁-C₆ alkyl, optionally substituted C₁-C₆ alkoxy, halo, and haloalkyl; and the subscript r is an integer from 0 to
 2. 15. The reaction mixture of claim 1, wherein the carbene precursor is a diazo reagent having a structure according to the formula:

wherein: R¹ is independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(1a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃; and R² is independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(2a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃; wherein R^(1a) and R^(2a) are independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl and -L-R^(C), wherein each L is selected from the group consisting of a bond, —C(R^(L))₂—, and —NR^(L)—C(R^(L))₂—, each R^(L) is independently selected from the group consisting of H, C₁₋₆ alkyl, halo, —CN, and —SO₂, and each R^(C) is selected from the group consisting of optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl; and each R⁷ and R⁸ is independently selected from the group consisting of H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl.
 16. The reaction mixture of claim 15, wherein R¹ and R² are both C₁ alkyl.
 17. The reaction mixture of claim 15, wherein the diazo reagent is selected from the group consisting of an α-diazoester, an α-diazoamide, an α-diazonitrile, an α-diazoketone, an α-diazoaldehyde, and an α-diazosilane.
 18. The reaction mixture of claim 17, wherein the diazo reagent has a formula selected from the group consisting of:

wherein R^(1a) is selected from the group consisting of H and optionally substituted C₁-C₆ alkyl; and each R⁷ and R⁸ is independently selected from the group consisting of H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl.
 19. The reaction mixture of claim 15, wherein the diazo reagent is selected from the group consisting of diazomethane, ethyl diazoacetate, and (trimethylsilyl)diazomethane.
 20. The reaction mixture of claim 1, wherein the olefinic substrate and the carbene precursor are part of the same substrate.
 21. The reaction mixture of claim 1, wherein the cyclopropanation product is produced in vitro.
 22. The reaction mixture of claim 1, wherein the reaction mixture further comprises a reducing agent.
 23. The reaction mixture of claim 1, wherein the heme enzyme is localized within a whole cell and the cyclopropanation product is produced in vivo.
 24. The reaction mixture of claim 1, wherein the cyclopropanation product is produced under anaerobic conditions.
 25. The reaction mixture of claim 1, wherein the heme enzyme is a variant thereof comprising a mutation at the axial position of the heme coordination site.
 26. The reaction mixture of claim 1, wherein the heme enzyme is a cyclochrome P450 enzyme or a variant thereof.
 27. The reaction mixture of claim 26, wherein the cyclochrome P450 enzyme is a P450 BM3 enzyme or a variant thereof.
 28. The reaction mixture of claim 27, wherein the P450 BM3 enzyme comprises the amino acid sequence set forth in SEQ ID NO:1.
 29. A method for producing a cyclopropanation product, the method comprising: (a) providing an olefinic substrate, a carbene precursor, and a heme enzyme; and (b) admixing the components of step (a) in a reaction for a time sufficient to produce a cyclopropanation product.
 30. The method of claim 29, wherein the cyclopropanation product is a compound according to Formula I:

wherein: R¹ is independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(1a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃; R² is independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR^(2a), C(O)N(R⁷)₂, C(O)R⁸, C(O)C(O)OR⁸, and Si(R⁸)₃; wherein R^(1a) and R^(2a) are independently selected from the group consisting of H, optionally substituted C₁₋₁₈ alkyl and -L-R^(C), wherein each L is selected from the group consisting of a bond, —C(R^(L))₂—, and —NR^(L)—C(R^(L))₂—, each R^(L) is independently selected from the group consisting of H, C₁₋₆ alkyl, halo, —CN, and —SO₂, and each Rc is selected from the group consisting of optionally substituted C₆₋₁₀ aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl; and R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H, C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, C₂₋₁₈ alkynyl, optionally substituted C₆₋₁₀ aryl, optionally substituted C₁-C₆ alkoxy, halo, hydroxy, cyano, C(O)N(R⁷)₂, NR⁷C(O)R⁸, C(O)R⁸, C(O)OR⁸, and N(R⁹)₂, wherein each R⁷ and R⁸ is independently selected from the group consisting of H, optionally substituted C₁₋₁₂ alkyl, optionally substituted C₂₋₁₂ alkenyl, and optionally substituted C₆₋₁₀ aryl; and each R⁹ is independently selected from the group consisting of H, optionally substituted C₆₋₁₀ aryl, and optionally substituted 6- to 10-membered heteroaryl, or two R⁹ moieties, together with the nitrogen atom to which they are attached, can form 6- to 18-membered heterocyclyl; or R³ forms an optionally substituted 3- to 18-membered ring with R⁴; or R⁵ forms an optionally substituted 3- to 18-membered ring with R⁶; or R³ or R⁴ forms a double bond with R⁵ or R⁶; or R³ or R⁴ forms an optionally substituted 5- to 6-membered ring with R⁵ or R⁶. 